Registration Dossier

Diss Factsheets

Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

Short term toxicity to fish

Short term toxicity toOryzias latipes(Himedaka) study was carried out for 48 hrs (Yashuhide TONOGAI et. al; 1982) .The study was based on the effects of the test compound (4 -(4 -aminophenyl)(4 -iminocyclohexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride (CAS no. 632 -99 -5) onOryzias latipesin a static fresh water system at a temperature of 25°C.Oryzias latipes(Himedaka) was used as a test organism. Test substance solution was prepared by dissolving the chemical in water and neutralized with 0.01 N NaOH or HCl, if necessary. Himedaka of the same age (about 2 cm in length, 0.2 g In weight) were chosen for the study. Test organismOryzias latipeswas acclimated for 10 days in the tap water before experiment. TLm test was carried out according to the procedure of Japan Industrial Standards. 10 fish of Himedaka per one trial were kept in 2 liter of deionized water at 25°C and, after 24 or 48 hrs, lethal concentration of 50% fish was determined. Based on effect on mortality of the test organismOryzias latipes, the 24 and 48 hrs LC50 value was determined to be 6.8 and 4.3 mg/l, respectively. Thus, based on the LC50 value, it can be concluded that the substance (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride cannot be classified in acute category as per the CLP classification criteria.

Long term toxicity to fish

Long term toxicity to Heteropneustes fossilis (Catfish) study was carried out for 60 days(American chemical society, 2017). The study was based on the effects of the test compound (4-(4-aminophenyl)(4-iminocyclohexa -2,5 -dienylidene)methyl)-2-methylaniline hydrochloride (CAS no. 632-99-5) on Heteropneustes fossilis in a brackish water system. Heteropneustes fossilis (Catfish) was used as a test organism. Test chemical concentration used for the study were 6.24 mg/l (for 96 h), 3.12 mg/l (for 10-20 days) and 1.56 mg/l (for 10 days), respectively. As no significant differences in the serum calcium and protein levels was observed from that of controls, the 60 days NOEC value was determined to be 3.12 mg/l, respectively.

Short term toxicity to aquatic invertebrates

Using the OECD QSAR toolbox version 3.3 (2017) with log kow as the primary descriptor and considering the five closest read across substances, the short term toxicity on aquatic invertebrates was predicted for target substance (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS no. 632 -99 -5). EC50 value was estimated to be 223 mg/l for Daphnia magna for 48 h duration. Based on this value it can be concluded that the substance (4 -(4 -aminophenyl)(4 -iminocyclo hexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride is considered to be non-toxic to aquatic environment and can be considered to be not classified as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Using the OECD QSAR toolbox version 3.3 (2017) with log kow as the primary descriptor and considering the five closest read across substances, the short term toxicity on aquatic algae and cyanobacteria was predicted for target substance (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS no. 632 -99 -5). EC50 value was estimated to be 450 mg/l for Pseudokirchneriella subcapitata for 72 h duration. Based on this value it can be concluded that the substance (4 -(4 -aminophenyl)(4 -iminocyclo hexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride is considered to be non-toxic to aquatic environment and can be considered to be not classified as per the CLP classification criteria.

Toxicity to micro-organisms

Toxicity to micro-organism study was carried out for48 hrs usingEscherichia colifor 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 onEscherichia coliwas determined to be 30 and 33 mg/l, respectively.

Additional information

Short term toxicity to fish

Various experimental key and supporting 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 short term toxicity to fish endpoint which were summarized as below:

 

In an experimental key study from peer reviewed journal (Yashuhide TONOGAI et. al; 1982), short term toxicity to Oryzias latipes (Himedaka) study was carried out for 48 hrs. The study was based on the effects of the test compound (4 -(4 -aminophenyl)(4 -iminocyclohexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride (CAS no. 632 -99 -5) on Oryzias latipes in a static fresh water system at a temperature of 25°C.Oryzias latipes (Himedaka) was used as a test organism. Test substance solution was prepared by dissolving the chemical in water and neutralized with 0.01 N NaOH or HCl, if necessary. Himedaka of the same age (about 2 cm in length, 0.2 g In weight) were chosen for the study. Test organism Oryzias latipes was acclimated for 10 days in the tap water before experiment. TLm test was carried out according to the procedure of Japan Industrial Standards. 10 fish of Himedaka per one trial were kept in 2 liter of deionized water at 25°C and, after 24 or 48 hrs, lethal concentration of 50% fish was determined. Based on effect on mortality of the test organism Oryzias latipes, the 24 and 48 hrs LC50 value was determined to be 6.8 and 4.3 mg/l, respectively.

 

Another short term toxicity to Heteropneustes fossilis (Catfish) study was carried out for 96 hrs (American chemical society, 2017). The study was based on the effects of the test compound (4 -(4 -aminophenyl)(4 -iminocyclohexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride(CAS no. 632 -99 -5) on Heteropneustes fossilis in a static brackish water system. Heteropneustes fossilis (Catfish) was used as a test organism. The 24, 48, 72 and 96 hrs LC50 value was determined to be 130.80, 36.50, 34.30 and 31.20 mg/l and the NOEC value was determined to be 0.5 mg/l respectively.

 

In a supporting study from peer reviewed journal (Bing Shen et. al; 2015) for the test chemical(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS No. 632-99-5), short term toxicity to Danio rerio (Zebra fish) study was carried out for 120 hrs. Adult Zebra fish AB strain was used as a test organism for the study. Zebrafish larvae were treated with dyes from 48 to 120 hpf for the acute toxicity assay, and from 72 to 120 hpf for the hepatotoxicity assay. They were housed in a light-and-temperature controlled aquaculture facility with a standard 14-h/10-h light/dark photoperiod and fed with live brine shrimp twice daily and dry flake once a day. Four to five pairs of zebrafish were set up for nature mating every time. On average, 200–300 embryos were generated. Embryos were maintained at 28 °C in fish water (0.2% Instant Ocean Salt in deionized water, pH 6.9-7.2, conductivity 480-510μS/cm and hardness 53.7-71.6 mg/l CaCO3). The embryos were washed and staged at 6 h post-fertilization (hpf) and 24 hpf. Five different test chemical concentrations were used for the study. They are 0.1, 1, 10, 100 and 1000 mg/l, respectively. Stock solutions were prepared in fish water and serial dilutions were made before each experiment. Zebrafish larvae were transferred to six-well plates, with 30 larvae per well in 3 ml of solution for the treatment period. The dissolved oxygen concentration in the solution was maintained above 80% throughout the experiments. Larvae treated with fish water were used as a vehicle control. To protect compounds from light-induced decomposition, experiments were carried out at a constant temperature (28 °C) in the dark. After treatment, zebrafish were observed and imaged with a dissecting stereomicroscope equipped with a high-speed video camera. Quantitative image analyzes were performed using image-based morphometric analysis. Mortality was recorded every 24 h. Dead zebrafish were defined as those lacking an observable heartbeat under a dissecting stereomicroscope. After treatments, all the zebrafish were killed with 0.25 g/l tricainemethanesulfonate, which conformed to the American Veterinary Medical Association (AVMA) requirements for killing by anesthetic. Mortality curves were generated using Origin 8.0 (OriginLab, USA). MNLC, LC10 and LC50 were estimated from this curve. Sigmoidal regression for concentration-response curves was used for estimation of MNLC, LC10 and LC50 (Origin 8.0). One-way ANOVA followed by Dunnett’s test was used to compare differences among the groups. All statistical analyzes were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) andP<0.05 was considered statistically significant. For quantitative analysis, all data were expressed as the mean ± standard error (SE). The incidence of abnormality in all groups treated with(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride were significantly greater than that in the vehicle control group. Dye also induced yolk sac absorption delay and swimming bladder deflation in a dose dependent manner. In addition, hepatotoxicity was observed in all groups treated with(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride. Based on the effect on mortality of the test organism Danio rerio (Zebra fish), the 120 hr NOEC, LC10, LC50 and LC100 value was determined to be 33.13, 43.47, 60.63 and approx. 102 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 and secondary source),it can be concluded that the test substance (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride cannot be classified in acute category as per the CLP classification criteria.

Long term toxicity to fish

Long term toxicity to Heteropneustes fossilis (Catfish) study was carried out for 60 days(American chemical society, 2017). The study was based on the effects of the test compound (4-(4-aminophenyl)(4-iminocyclohexa -2,5 -dienylidene)methyl)-2-methylaniline hydrochloride (CAS no. 632-99-5) on Heteropneustes fossilis in a brackish water system. Heteropneustes fossilis (Catfish) was used as a test organism. Test chemical concentration used for the study were 6.24 mg/l (for 96 h), 3.12 mg/l (for 10-20 days) and 1.56 mg/l (for 10 days), respectively. As no significant differences in the serum calcium and protein levels was observed from that of controls, the 60 days NOEC value was determined to be 3.12 mg/l, respectively.

Short term toxicity to aquatic invertebrates

Various predicted data for the target chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS No. 632-99-5) and various supporting weight of evidence studies for its closest read across substances with logKow as the primary descriptorwere reviewed to summarize the following information:

 

Short term toxicity on aquatic invertebrate of target chemical(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS No. 632-99-5) is predicted using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances (2017). On the basis of effects observed in a static freshwater system, the effect concentration EC50 value for the substance is estimated to be 233 mg/l for Daphnia magna for 48 hr duration.Based on this value it can be concluded that the substance (4 -(4 -aminophenyl)(4 -iminocyclo hexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride is considered to be non-toxic to aquatic environment and can be considered to be not classified as per the CLP classification criteria.

 

In another prediction done using the EPI Suite ECOSAR version 1.10 (EPI Suite, 2017), the short term toxicity on aquatic invertebrates was predicted for test substance(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS No. 632-99-5).On the basis of effects observed in a static freshwater system, the effect concentration EC50 value for the substance is estimated to be 1186.437 mg/l for Daphnia magna for 48 h duration.Based on this value it can be concluded that the substance (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride is considered to be not toxic to aquatic environment and can be not classified as per the criteria mentioned in CLP regulation.

 

In a weight of evidencestudy from authoritative database (ECOTOX database, 2017) of the read across chemical 4-[(4-dimethylaminophenyl)-phenylmethylidene]cyclohexa-2,5-dien-1-ylidene]-dimethylazanium chloride (CAS no. 569-64-2),Short term toxicity to Litopenaeus schmitti (Southern white shrimp) study was carried out for 48 hrs. The study was based on the effects of the read across compound 4 -[(4 -dimethylamino phenyl)-phenylmethylidene]cyclohexa-2,5-dien-1-ylidene]-dimethylazanium chloride on Litopenaeus schmitti in a static salt water system. Litopenaeus schmitti (Southern white shrimp) was used as a test organism. Based on effect on mortality of the test organism Litopenaeus schmitti, the 48 hr LC50 value was determined to be 77170 mg/l (LC50 value ranges from 60470 to 109120 mg/l), respectively. Thus, based on the LC50 value, it can be concluded that the substance 4 -[(4 -dimethylaminophenyl)-phenylmethyli dene]cyclohexa-2,5-dien-1-ylidene]-dimethylazanium chloride can be considered as non-toxic to aquatic organisms and thus can be considered to be not classified as per the CLP classification criteria.

 

Another short term toxicity to Daphnia magna (Water flea) studyof the read across chemical 1-Hexene (CAS no. 592-41-6)was carried out for 48 hrs(ECOTOX database, 2017). The study was based on the effects of the read across compound 1-Hexene on Daphnia magna in a static fresh water system at a temperature of 20⁰C, hardness of water was approx. 210 mg/l CaCO3, dissolved oxygen ≥ 70%and pH range of 8.0 to 8.2, respectively. Daphnia magna (Water flea) of < 24 h old was used as a test organism. Study was performed using synthetic medium in glass stopped conical flask. Based on effect on intoxication of the test organism Daphnia magna, the 48 hr EC50 value was determined to be 30 mg/l. Thus, based on the EC50 value, it can be concluded that the substance 1-Hexenecan be considered as toxic to aquatic organisms. Since the test chemical 1-Hexene is readily biodegradable in nature, chemical 1-Hexene can be considered as non-toxic to aquatic organism and thus cannot be classified as hazardous as per the CLP classification criteria.

 

For the same read across chemical 1-Hexene(CAS no. 592-41-6) from authoritative database (ECOTOX database, 2017), short term toxicity to Daphnia magna (Water flea) study was carried out for 48 hrs. The study was based on the effects of the test compound1-Hexene(CAS no. 592-41-6) on Daphnia magna in a static fresh water system at a temperature of 20⁰C, hardness of water was approx. 210 mg/l CaCO3, dissolved oxygen ≥ 70% and pH range of 8.0 to 8.2, respectively. Daphnia magna (Water flea) of< 24 h old was used as a test organism. Study was performed using synthetic medium in glass beaker covered with a watch glass. Based on effect on intoxication of the test organism Daphnia magna, the 48 hr EC50 value was determined to be 60 mg/l (EC50 value ranges from > 32 to < 100 mg/l, respectively).Thus, based on the EC50 value, it can be concluded that the substance 1-Hexene can be considered as toxic to aquatic organisms. Since the test chemical 1-Hexene is readily biodegradable in nature, chemical 1-Hexene can be considered as non-toxic to aquatic organism and thus cannot be classified as hazardous as per the CLP classification criteria.

 

In a supporting weight of evidence study from secondary source (OECD SIDS, 2001), short term toxicity to Daphnia magna (Water flea) study was carried out for 48 hrs. The study was based on the effects of the read compound 1-Hexene (CAS no. 592-41-6) on Daphnia magna in a static fresh water system. The 24 and 48 hrs EC50 value was determined to be 540 and 230 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance 1-Hexene can be considered as non-toxic to aquatic organism and thus cannot be classified as hazardous as per the CLP classification criteria.

 

Thus, based on the overall reported results for target chemical(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride(OECD QSAR toolbox version 3.3 and EPI suite, 2017) and for its read across substance (fromauthoritative database ECOTOX and secondary source OECD SIDS),it can be concluded that the test substance (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride can be considered as non-toxic to aquatic environment and can be considered to be not classified as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Various predicted data for the target chemical (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS No. 632-99-5) and various supporting weight of evidence studies for its closest read across substances with logKow as the primary descriptorwere reviewed to summarize the following information:

 

Short term toxicity on aquatic algae and cyanobacteria of target chemical(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS No. 632-99-5) is predicted using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances (2017). On the basis of effects observed in a static freshwater system, the effect concentration EC50 value for the substance is estimated to be 450 mg/l for Pseudokirchneriella subcapitata for 72 hr duration.Based on this value it can be concluded that the substance (4 -(4 -aminophenyl)(4 -iminocyclo hexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride is considered to be non-toxic to aquatic environment and can be considered to be not classified as per the CLP classification criteria.

 

In another prediction done using the EPI Suite ECOSAR version 1.10 (EPI Suite, 2017), the short term toxicity on green algae was predicted for test substance3(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride (CAS No. 632-99-5).On the basis of effects observed in a static freshwater system, the effect concentration EC50 value for the substance is estimated to be 599 mg/l for green algae for 96 h duration.Based on this value, it can be concluded that the test chemical (4 -(4 -aminophenyl)(4 -iminocyclo hexa-2,5 -dienylidene)methyl)-2-methylaniline hydrochloride can be considered as non-toxic to green algae at environmentally relevant concentrations and can be considered not classified as per the CLP classification criteria.

 

In a weight of evidencestudy from secondary source (OECD SIDS, 2014 and High Production Volume (HPV) Challenge Program; 2017) of the read across chemical 1-Octadecene (CAS no. 112-88-9), short term toxicity to Selenastrum capricornutum study was carried out for 96 hrs. The study was performed according to OECD Guideline 201 (Alga, Growth Inhibition Test).The study was based on the effects of the read across compound1-Octadecene(CAS no. 112-88-9) on Selenastrum capricornutum in a static fresh water system at a temperature range of 22 to 26°C and pH of 7.4 to 7.7, respectively. Test chemical concentration used for the study were1.0, 2.2, 4.6, 10, 22, 46, 100, 220, 460 and 1000 mg/L (nominal concentrations), respectively. Analar Acetone was used as a vehicle for the study. Selenastrum capricornutum ATCC 22662 was used as a test organism for the study obtained from the axenic laboratory culture derived from a strain obtained from the American Type Culture Collection (Maryland, USA).Sixteen Erlenmeyer flasks containing 50 ml of culture medium were prepared. The remaining six flasks received no 1-Octadecene, however, acetone concentrations in all flasks (including the controls) were adjusted to 0.1 mg/L. Each flask was inoculated with S. capricornutum to an initial concentration of 500 cells/ml. Flasks were incubated at 100 cycles/min under constant illumination (approximately 3000 lux). Tests temperatures ranged from 22-26°C and pH of test solutions ranged from 7.4-7.7 s.u. None of the concentrations of the test chemical tested caused a reduction in cell number at day 4 compared to the mean cell number at day 4 in the controls. Therefore, the 96 hr NOEC and EC50 value was determined to be 1000 and >1000 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance 1-Octadecene can be considered as non-toxic to aquatic organism and thus cannot be classified as hazardous as per the CLP classification criteria.

 

For the read across chemical 1-Hexene(CAS no. 592-41-6), short term toxicity to green algae study was carried out for 96 hrs (OECD SIDS, 2001). The study was based on the effects of the read across compound 1-Hexene on green algae in a static fresh water system. The 96 hr EC50 value was determined to be 22 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance1-Hexene can be considered as toxic to aquatic organisms. Since the test chemical 1 -Hexene is readily biodegradable in nature, chemical 1 -Hexene can be considered as non-toxic to aquatic organism and thus cannot be classified as hazardous as per the CLP classification criteria.

 

Thus, based on the overall reported results for target chemical(4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride(OECD QSAR toolbox version 3.3 and EPI suite, 2017) and for its read across substance (fromsecondary source),it can be concluded that the test substance (4 -(4 -aminophenyl)(4 -iminocyclohexa-2,5 -dienylidene)methyl)-2 -methylaniline hydrochloride can be considered as non-toxic to aquatic environment and can be considered to be notclassified as per the CLP classification criteria.

Toxicity to micro-organisms

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.