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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
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:
Short term toxicity to Oryzias latipes (Himedaka) study was carried out for 48 hrs.
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: Fuschine
- 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
- Other: Test chemical used was of commercial products of guaranteed grade.
Analytical monitoring:
not specified
Details on sampling:
- Sampling method: Test substance solution was prepared by dissolving the chemical in water and neutralized with 0.01 N NaOH or HCl, if necessary.
Vehicle:
not specified
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Common name: Himedaka
- Age at study initiation (mean and range, SD): Exact age of the test organism was not mentioned, but the no. of animals used for the study was of same age.
- Length at study initiation (length definition, mean, range and SD): Length of the test organism was 2 cm.
- Weight at study initiation (mean and range, SD): 0.2 g

ACCLIMATION
- Acclimation period: 10 days
- Acclimation conditions (same as test or not): Test organism Oryzias latipes was acclimated for 10 days in the tap water before experiment.
Test type:
static
Water media type:
freshwater
Total exposure duration:
48 h
Test temperature:
25°C
Details on test conditions:
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.
Reference substance (positive control):
not specified
Key result
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
6.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
4.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Sublethal observations / clinical signs:

Table: TLm values of test chemical Fuchsine (dyestuff).

 

 

Substance

TLm (mg/l)a

24 hr

48 hr

Fuchsine

6.8

4.3

 

Where,

a = Ten fish of Himedaka were used in 2 liter of each solution. 

Validity criteria fulfilled:
not specified
Conclusions:
For chemical, (4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride, based on effect on mortality of the test organism Oryzias latipes (Himedaka), the 24 and 48 hrs LC50 value was determined to be 6.8 and 4.3 mg/l, respectively.
Executive summary:

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. 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.

Description of key information

Short term toxicity to Oryzias 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.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
4.3 mg/L

Additional information

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.