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Reference
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
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-organism study was carried out using Escherichia coli for determining the bacteriostatic effect of test chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (Magenta I).
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (IUPAC name): (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride
- Common name: Rosanilin chloride (Magenta I)
- Molecular formula: C20H19N3.HCl
- Molecular weight: 337.852
- Smiles notation: C(\c1cc(c(N)cc1)C)(c1ccc(N)cc1)=C1/C=CC(=N)C=C1.Cl
- InChl: 1S/C20H19N3.ClH/c1-13-12-16(6-11-19(13)23)20(14-2-7-17(21)8-3-14)15-4-9-18(22)10-5-15;/h2-12,21H,22-23H2,1H3;1H/b20-14-,21-17?;
- Substance type: Organic
- Physical state: Solid
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Escherichia coli
Details on inoculum:
- Laboratory culture: Escherichia coli isolated from the feces was used as a test organism for the study.
- Method of cultivation: The organism was grown until well established in the test medium.
- Preparation of inoculum for exposure: For the study, a young culture in the logarithmic phase of growth was so diluted that the amount used for each inoculation contained approx. 100 organisms.
Test type:
not specified
Water media type:
not specified
Total exposure duration:
48 h
Test temperature:
37⁰C
pH:
6.9
Nominal and measured concentrations:
Nominal concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: Test tubes were used as a test vessel for the study.
- No. of organisms per vessel: Each test vessel contains approx. 100 organisms
- No. of vessels per concentration (replicates): Duplicate
- Biomass loading rate: For the study, a young culture of test organism E. coli was so diluted that the amount used for each inoculation contained approx. 100 organisms.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: The test medium contains 1% peptone, 1% lactose to which varying amounts of aqueous solution of test chemical have been added.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Visible growth of test chemical was examined after 24 and 48 hours of study. Results were recorded in terms of least concentration of the test chemical which will inhibit growth.
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: No growth inhibition of test organism E. coli was observed.
Remarks on result:
other: Dilution factor is 1: 32,500 (0.003%)
Key result
Duration:
48 h
Dose descriptor:
other: MIC
Effect conc.:
33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: Dilution factor is 1: 30,000 (0.0033%)

Table: Bacteriostatic action of Magenta I against the test organism Escherichia col.

 

Designation of dye

 

Rosanilin (Magenta I)

Dilution of dye

 

1: 10,000

1: 12,000

1: 14,000

1: 16,000

1: 18,000

1: 20,000

1: 30,000

1: 32,500

1: 35,000

1: 45,000

1: 47,500

1: 50,000

1: 52,500

1: 55,000

 

++

++

++

++

++

++

++

- -

- -

- -

- -

- -

- -

- -

 

 

 

Where,

+ = indicate bacteriostasis (no growth of bacteria)

-         = indicate growth.

Duplicate columns indicate duplicate tubes.

Tests in lactose-peptone broth, pH 6.9; incubation at 37⁰C for 48 hrs.

Validity criteria fulfilled:
not specified
Conclusions:
The 48 hrs NOEC and MIC value for test chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride on Escherichia coli was determined to be 30 and 33 mg/l, respectively.
Executive summary:

Toxicity to micro-organism study was carried out for48 hrs using Escherichia coli for determining the bacteriostatic effect of test chemical (4-(4-aminophenyl)(4-imino cyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (Magenta I) (CAS no. 632-99-5). Escherichia coli isolated from the feces was used as a test organism for the study. The test medium contains 1% peptone, 1% lactose to which varying amounts of aqueous solution of test chemical have been added. The organism was grown until well established in the test medium. For the study, a young culture in the logarithmic phase of growth was so diluted that the amount used for each inoculation contained approx. 100 organisms. Duplicate tubes were made and incubated at 37⁰C for a period of 48 hrs. Visible growth of test chemical was examined after 24 and 48 hours of study. Results were recorded in terms of least concentration of the test chemical which will inhibit growth. The 48 hrs NOEC and MIC value for test chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride on Escherichia coli was determined to be 30 and 33 mg/l, respectively.

Description of key information

Toxicity to micro-organism study was carried out for48 hrs using Escherichia coli for determining the bacteriostatic effect of test chemical (4-(4-aminophenyl)(4-imino cyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (Magenta I) (CAS no. 632-99-5) (Cassandra Ritter et. al; 1940). Escherichia coliisolated from the feces was used as a test organism for the study. The test medium contains 1% peptone, 1% lactose to which varying amounts of aqueous solution of test chemical have been added. The organism was grown until well established in the test medium. For the study, a young culture in the logarithmic phase of growth was so diluted that the amount used for each inoculation contained approx. 100 organisms. Duplicate tubes were made and incubated at 37⁰C for a period of 48 hrs. Visible growth of test chemical was examined after 24 and 48 hours of study. Results were recorded in terms of least concentration of the test chemical which will inhibit growth. The 48 hrs NOEC and MIC value for test chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride on Escherichia coli was determined to be 30 and 33 mg/l, respectively.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
30 mg/L

Additional information

Various experimental weight of evidence studies for the target chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS No. 632-99-5)were reviewed for toxicity to micro-organisms endpoint which were summarized as below:

 

In an experimental study from peer reviewed journal (Cassandra Ritter et. al; 1940), toxicity to micro-organism study was carried out for48 hrs using Escherichia coli for determining the bacteriostatic effect of test chemical (4-(4-aminophenyl)(4-imino cyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (Magenta I) (CAS no. 632-99-5). Escherichia coli isolated from the feces was used as a test organism for the study. The test medium contains 1% peptone, 1% lactose to which varying amounts of aqueous solution of test chemical have been added. The organism was grown until well established in the test medium. For the study, a young culture in the logarithmic phase of growth was so diluted that the amount used for each inoculation contained approx. 100 organisms. Duplicate tubes were made and incubated at 37⁰C for a period of 48 hrs. Visible growth of test chemical was examined after 24 and 48 hours of study. Results were recorded in terms of least concentration of the test chemical which will inhibit growth. The 48 hrs NOEC and MIC value for test chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride on Escherichia coli was determined to be 30 and 33 mg/l, respectively.

 

Another supporting weight of evidence toxicity to micro-organism study was carried out for 18 to 24 hrs at a temperature of 37⁰C using Staphylococcus aureus for determining the MIC of test chemical (4 -(4 -aminophenyl)(4 -iminocyclohexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride (CAS no. 632-99-5) (Tomoko Fujiyuki et. al; 2010). Staphylococcus aureus was isolated from clinical samples. A clinical strain of S. aureus, MSSA1, was cultured in Luria-Bertani 10 (LB 10) medium (10 g bactotryptone, 5 g yeast extract, 10 g NaCl per liter) at 37°C for 24 h. The full growth culture was diluted to 1/1,000 in Mueller-Hinton (MH) medium and then a 100-μL aliquot was added to each well of 96-well plate. Test chemical fuchsin basic was dissolved in phosphate buffered saline (PBS, 10 mM sodium phosphate, 137 mM sodium chloride, 3 mM potassium chloride, pH 7.4) or dimethyl sulfoxide. Bacterial growth was visually determined. The MIC (minimum inhibitory concentration) was defined as the lowest concentration of the dye that inhibited the bacterial growth. The minimum inhibitory concentration of the test chemical (4 -(4 -aminophenyl)(4 -iminocyclohexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride against Staphylococcus aureus was determined to be 6 mg/l.

 

In an supporting weight of evidence study from peer reviewed journal (Auriol C . Hilll; 1985), toxicity to micro-organism study was carried out using different mycoplasmas spp. using the microtitre-plate method for determining the bacteriostatic effect of test chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (Magenta I) (CAS no. 632-99-5). Test chemical concentration ranges from 0.5% to 0.000005%, respectively. Chemical basic fuchsin was first dissolved in ethanol and then diluted in distilled water. For the dye sensitivity tests, these stock solutions were further diluted in growth medium to give a range of final concentrations between 0.5% and 0.000005%. Mycoplasma type cultures were obtained from the National Collection of Type Cultures, LondonNW9,UK and other laboratories, respectively. Some unidentified primate mycoplasmas were received from Drs B. C. Cole (University of Utah, Utah, USA) and others were isolated at the former MRC Laboratory Animals Centre from animals sent for diagnosis of disease or for microbiological screening. Depending upon their biochemical activity mycoplasmas were grown in basic liquid medium containing either 1% (w/v) glucose, pH 7.8 or 1%(w/v) arginine,pH7.3.: Aliquots of cultures were stored in sealed ampoules at -70⁰C.A drop (0.05 ml) of the appropriate medium (without phenol red) was deposited in each of a row of wells in a microtitre plate. The stock solution of the dye under test (0.05 ml) was added to the first well and doubling dilutions were made to well 12. Medium (0.1 ml) was added to each well, followed by 045 ml of the mycoplasma suspension at a dilution calculated to produce a pH change in 3 to5d in control medium wells containing phenol red but no dye. Plates were sealed and incubated at 35"Cand examined for growth from each well. When the control well showed a pH change of half a unit (or a distinct colour change in medium containing resazurin) viability was examined by inoculation of a standard drop(0,025ml) of suspension taken from each well, including controls, onto an agar plate (visual estimation of growth, as in conventional metabolism tests. was precluded by the presence of dyes). Plates were incubated and examined for growth from each well. Statistical analysis involve the calculation of log of the dye inhibition titre. Effective concentration of test chemical on the growth of the mycoplasmas spp. was determined to be ranges from 0.6 to 300 mg/l, respectively.

 

Thus, based on the overall reported results for target chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (from peer reviewed journals), it can be concluded that the MIC value for the test substance (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride determined to be 6 & 33 mg/l and EC valueranges from 0.6 to 300 mg/l, respectively.