N-ethyl-2-(isopropyl)-5-methylcyclohexanecarboxamide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

as mentioned below

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

WoE report is based on toxicity study of microorganism for the test chemical :
2.Toxicity of menthone on the microorganisms Pseudomonas citronellolis DSM 50332 was studied for 21 days.
3.The antibacterial activities of twenty-one oxygenated monoterpenes including test chemical was determine against 63 bacterial strains by disc diffusion method.

Details on sampling:

1) DETAILS ON PRETREATMENT
- Centrifugation: no data
- Filtration: no data
- Digestion (acid used, method: e.g. micro-oven): no data
- Extraction (solvent used, method: e.g. liquid-liquid, SPE): no data
- Clean up method:e.g. chemical used for chemistry method (Cu, Hg, ...) or phase and solvent used for SPE method: no data
- Derivatisation method if used: no data
- Concentration (method): no data

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
- Separation method (e.g. HPLC, GC): Nitrate and nitrite were separated by high-performance liquid chromatography (HPLC) by using a type A09 micro anion-exchange column
- Conditions (column, mobile phase, etc.): Transformation products extracted into the hexane phase were separated on a type NB-54 column (0.32 mm by 25 m; Nordion, Helsinki, Finland) by using a temperature gradient (the injection port and transfer line temperature was 2508C, the initial column temperature was 508C, and the column temperature was increased to 2008C at a rate of 58C min21) and helium as the carrier gas.

- Detection method (e.g. ECD, UV, MS, ICP-AES, ICP-MS): Monoterpene contents were determined with a gas chromatograph equipped with flame ionization detectors and connected to a digital data-analyzing system
- Detection limits (LOD, LOQ) (indicate method of determination/calculation): no data
- Reproducibility in % (indicate method of evaluation; should be given for stated concentration levels): no data
- Linearity range: no data
- Internal or external calibration: no data
- Extraction recovery (indicate if results are corrected or not for recoveries):
- Method of confirmation of identity of measured compound: Compounds were identified by comparing them with injected standards, compounds in the library at the National Institute of Standards and Technology, and Compounds in a terpene library.

Vehicle:

yes

Remarks:

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):methanol - Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):1 ml o

Test organisms (species):

other: 2).Pseudomonas citronellolis DSM 50332 , 3)Clavibacter michiganense,Xanthomonas campestrispv. rhapontici,Klebsiella trevisanii,Pseudomonas aeruginosa ATCC 9027

Details on inoculum:

2) - Laboratory culture: Pseudomonas citronellolis DSM 50332 was obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany.

- Name and location of sewage treatment plant where inoculum was collected: Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany.

- Method of cultivation: no data

- Preparation of inoculum for exposure: Pure cultures were isolated anaerobically on agar bottle plates in which the substrate was provided by diffusion from a reservoir through the gas phase to the agar surface (10, 19). During all manipulations, the bottles containing the agar were flushed with N2-CO2 (90:10, vol/vol) by using a Hungate syringe. A 20-ml portion of liquid anoxic medium containing 1.5% washed agar was prepared with chelated trace elements instead of non-chelated trace elements (20) and with 50 mM carbonate instead of the phosphate-carbonate buffer above, dispensed into a 100-ml bottle, and solidified quickly by placing the bottle on a plastic bag filled with ice. After inoculation, the agar bottles were inverted, and 75 ml of monoterpene in 3 ml of HMN was added to each bottle. During incubation, the bottles were kept horizontal with the agar aloft. Direct contact between the organic phase and the agar was avoided

- Pretreatment: no data

- Initial biomass concentration: no data

Test type:

static

Water media type:

other: 2. Agar diffusion method.

Total exposure duration:

21 d

Remarks on exposure duration:

2) Test conducted for 10 days to 21 days.

pH:

7

Nominal and measured concentrations:

10 μl

Details on test conditions:

2) - Test vessel: Glass bottels
- Material, size, headspace, fill volume: 0.5 liter of glass bottles that contained 400 ml of medium, 4 ml of HMN, and 200 ml of liquid substrate

- Biomass loading rate: 0.5-liter bottles that contained 400 ml of medium, 4 ml of HMN, and 200 ml of liquid substrate or 200 mg of solid substrate under an N2-CO2 (90:10, vol/vol). A 100-ml portion of the water-mud mixture from the ditch was inoculated into each bottle together with 350 ml of medium, 8 ml of HMN, and 400 ml or 400 mg of substrate.

- Photoperiod: performed in dark

3)- Laboratory culture: Bacterial cultures were preserved in Luria Broth and 15% glycerol solution at -80 deg.C prior to use.
- source of Microorganisms: Microorganisms were provided from the Department of Clinical Microbiology, Faculty of Medicine and Plant Diagnostic Laboratory,Faculty of Agriculture, Ataturk University,Erzurum, Turkey.
- Method of cultivation:in petri dish
- Preparation of inoculum for exposure:Suspensions (100 μl) of the bacteria, adjusted to 108 cfu/ml final cell concentration, were added to flasks containing 25 ml sterile NA medium and then poured into Petri dishes and spread by a sterile swab (9 cm). 30 mg of each of the compounds were dissolved in 1 ml of methanol and these solutions were sterilized in 0.45 μm milipore filters. Sterilized discs (5 mm) were soaked with 10 μl of each compound solution. These discs were put in the middle of plates containing NA medium.
Penicillin was used as a positive control. For this purpose, 1 mg of penicillin was added into 1 ml sterilized water, and a sterilized disc was soaked with 10 μl of this solution. Bacterial cultures of plant origins were incubated at (27 deg.C), whereas the bacterial cultures of clinic and food origins were incubated at (35 Deg.C) for 6 d. At the end of six-day-periods, inhibition zones were measured in mm. All the tests were made in triplicate.

Suspensions (100 μl) of the bacteria, adjusted to 108 cfu/ml final cell concentration, were added to flasks containing 25 ml sterile NA medium and then poured into Petri dishes and spread by a sterile swab (9 cm). 30 mg of each of the compounds were dissolved in 1 ml of methanol and these solutions were sterilized in 0.45 μm milipore
filters. Sterilized discs (5 mm) were soaked with 10 μl of each compound solution. These discs were put in the middle of plates containing NA medium.
Penicillin was used as a positive control.
For this purpose, 1 mg of penicillin was added into 1 ml sterilized water, and a sterilized disc was soaked with 10 μl of this solution. Bacterial cultures of plant origins were incubated at (27 deg.C), whereas the bacterial cultures of clinic and food origins were incubated at (35 deg.C) for 6 d. At the end of six-day-periods,inhibition zones were measured in mm. All the tests were made in triplicate.

Reference substance (positive control):

yes

Remarks:

2) Pasteurized samples and inoculated preparations without nitrate or monoterpene were used as controls.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2 other: mM

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of anaerobic gas production

Remarks on result:

other: No inhibition was observed

Duration:

6 d

Dose descriptor:

other: MIC

Effect conc.:

10 other: μl

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: showed effects on 4 bacterial strain out of 63 strain tested.

Validity criteria fulfilled:

not specified

Conclusions:

Test chemical has no adverse effects on microorganisms

Executive summary:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the effect of test material on microorganisms.The studies are as mentioned below:

For the structurally similar read across substance toxicity of menthone on the growth of microorganismsPseudomonas citronellolisDSM 50332 for 21 days. Analytical grade samples were used for the test. Test performed in 0.5 liter of agar bottle plates by using the pure culture of microorganism providing proper temperature and PH. Culture were inoculated in dark having the mesophilic temperature. Growth inhibition ofPseudomonas citronelloliswas observed at 2m M of concentration of menthone for minimum 10 days to 3 weeks. Analytical grade samples were used for the test. Thus after the exposure with chemical no inhibition was observed. On the basis of no growth inhibition of microorganismsPseudomonas citronellolisdue to the menthone for 10 days to 3 weeks, the NOEC was 2mM.

In another data for structrually similar read across substance , the objective of this study was to evaluate the inhibitory effects of 21 pure oxygenated monoterpenes, obtained commercially including test chemical on the growth of 63 bacterial strains (plant, food and clinic origins). In experiment Suspensions (100μl) of the bacteria, adjusted to 108 cfu/ml final cell concentration, were added to flasks containing 25 ml sterile NA medium and then poured into Petri dishes and spread by a sterile swab (9 cm). 30 mg of each of the compounds were dissolved in 1 ml of test chemical and these solutions were sterilized in 0.45μm milipore filters. Sterilized discs (5 mm) were soaked with 10μl of each compound solution. These discs were put in the middle of plates containing NA medium. Penicillin was used as a positive control. For this purpose, 1 mg of penicillin was added into 1 ml sterilized water, and a sterilized disc was soaked with 10μl of this solution. Bacterial cultures of plant origins were incubated at (27 ± 2) °C, whereas the bacterial cultures of clinic and food origins were incubated at (35 ± 2) °C for 6 d. At the end of six-day-periods, inhibition zones were measured in mm. All the tests were made in triplicate. At end of the experiment the test substance showed inhibitory effects on growth of the 4 bacterial strains (Clavibacter michiganense, Xanthomonas campestrispv.Rhapontici,Klebsiella trevisanii,Pseudomonas aeruginosa ATCC 9027) out of 63 strains at a concenttration of 10μl. Test chemical has no adverse effects on microorganisms

Description of key information

Toxicity to microorganisms:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the effect of test material on microorganisms.The studies are as mentioned below:

For the structurally similar read across substancetoxicity of menthone on the growth of microorganismsPseudomonas citronellolisDSM 50332 for 21 days. Analytical grade samples were used for the test. Test performed in 0.5 liter of agar bottle plates by using the pure culture of microorganism providing proper temperature and PH. Culture were inoculated in dark having the mesophilic temperature. Growth inhibition ofPseudomonas citronelloliswas observed at 2m M of concentration of menthone for minimum 10 days to 3 weeks. Analytical grade samples were used for the test. Thus after the exposure with chemical no inhibition was observed. On the basis of no growth inhibition of microorganismsPseudomonas citronellolisdue to the menthone for 10 days to 3 weeks, the NOEC was 2mM.

In another data for structrually similar read across substance , the objective of this study was to evaluate the inhibitory effects of 21 pure oxygenated monoterpenes, obtained commercially including test chemical on the growth of 63 bacterial strains (plant, food and clinic origins). In experiment Suspensions (100μl) of the bacteria, adjusted to 108 cfu/ml final cell concentration, were added to flasks containing 25 ml sterile NA medium and then poured into Petri dishes and spread by a sterile swab (9 cm). 30 mg of each of the compounds were dissolved in 1 ml of test chemical and these solutions were sterilized in 0.45μm milipore filters. Sterilized discs (5 mm) were soaked with 10μl of each compound solution. These discs were put in the middle of plates containing NA medium. Penicillin was used as a positive control. For this purpose, 1 mg of penicillin was added into 1 ml sterilized water, and a sterilized disc was soaked with 10μl of this solution. Bacterial cultures of plant origins were incubated at (27 ± 2) °C, whereas the bacterial cultures of clinic and food origins were incubated at (35 ± 2) °C for 6 d. At the end of six-day-periods, inhibition zones were measured in mm. All the tests were made in triplicate. At end of the experiment the test substance showed inhibitory effects on growth of the 4 bacterial strains (Clavibacter michiganense, Xanthomonas campestrispv.Rhapontici,Klebsiella trevisanii,Pseudomonas aeruginosa ATCC 9027) out of 63 strains at a concenttration of 10μl.Test chemical has no adverse effects on microorganisms

Additional information

Toxicity to microorganisms:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the effect of test material on microorganisms.The studies are as mentioned below:

For the structurally similar read across substancetoxicity of menthone on the growth of microorganismsPseudomonas citronellolisDSM 50332 for 21 days. Analytical grade samples were used for the test. Test performed in 0.5 liter of agar bottle plates by using the pure culture of microorganism providing proper temperature and PH. Culture were inoculated in dark having the mesophilic temperature. Growth inhibition ofPseudomonas citronelloliswas observed at 2m M of concentration of menthone for minimum 10 days to 3 weeks. Analytical grade samples were used for the test. Thus after the exposure with chemical no inhibition was observed. On the basis of no growth inhibition of microorganismsPseudomonas citronellolisdue to the menthone for 10 days to 3 weeks, the NOEC was 2mM.

In another data for structrually similar read across substance , the objective of this study was to evaluate the inhibitory effects of 21 pure oxygenated monoterpenes, obtained commercially including test chemical on the growth of 63 bacterial strains (plant, food and clinic origins). In experiment Suspensions (100μl) of the bacteria, adjusted to 108 cfu/ml final cell concentration, were added to flasks containing 25 ml sterile NA medium and then poured into Petri dishes and spread by a sterile swab (9 cm). 30 mg of each of the compounds were dissolved in 1 ml of test chemical and these solutions were sterilized in 0.45μm milipore filters. Sterilized discs (5 mm) were soaked with 10μl of each compound solution. These discs were put in the middle of plates containing NA medium. Penicillin was used as a positive control. For this purpose, 1 mg of penicillin was added into 1 ml sterilized water, and a sterilized disc was soaked with 10μl of this solution. Bacterial cultures of plant origins were incubated at (27 ± 2) °C, whereas the bacterial cultures of clinic and food origins were incubated at (35 ± 2) °C for 6 d. At the end of six-day-periods, inhibition zones were measured in mm. All the tests were made in triplicate. At end of the experiment the test substance showed inhibitory effects on growth of the 4 bacterial strains (Clavibacter michiganense, Xanthomonas campestrispv.Rhapontici,Klebsiella trevisanii,Pseudomonas aeruginosa ATCC 9027) out of 63 strains at a concenttration of 10μl.Test chemical has no adverse effects on microorganisms