Registration Dossier

Administrative data

Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study without detailed documentations.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-methylaniline (100-61-8)
- Physical state: solved in reconstituted water
- Analytical purity: reagent grade
- Supplier: Merck, 6100 DarmstacE/FR Germany

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
No details specified.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Brachydanio rerio
- Source: West Aquarium, 3/422 Bad Lauterberg/FR Germany

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish to induce spawning: 4 females, 8 males
- Method of collection of fertilised eggs: the spawn was collected on the bottom of a glass box covered by a steel wire-net containing some small green glass trees
- Subsequent handling of eggs: the eggs were separated and cultured at 25°C individually using micro plates (Nunc, Roshilde/Denmark) containing 100 µL reconstituted water.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
Until hatching.
Post exposure observation period:
Not applicable.

Test conditions

Hardness:
200 mg/L
Test temperature:
25°C
pH:
8 ± 0.2
Dissolved oxygen:
No data.
Salinity:
2 mM CaCI₂.2 H.O, 0.078 mM KCI, 0.5 mM MgSO₄.7 H₂O, 0.77 mM NHCO₃, in demineralized water
Nominal and measured concentrations:
7 concentrations between 0.1-5 µmol/L
Details on test conditions:
TEST SYSTEM
- No. of fertilized eggs/embryos per vessel: 12
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

EFFECT PARAMETERS MEASURED: marked signs of development, e.g. abnormalities, and deaths during the 96 h test period.

Reference substance (positive control):
not required

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.2 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Oedematous enlargement of the pericardial space with retardation of heart development and reduced blood flow , deformation of the skeleton and the muscle apparatus and general retardation of the embryo development.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.71 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 0.69-0.72
Duration:
96 h
Dose descriptor:
other: LC100
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Mortality/survival at embryo and larval stages:
- no deaths or morphological changes were observed in the controls
- an obvious association excisted between malformations and death
Type of morphological abnormalities:
- oedematous enlargement of the pericardial space with retardation of heart development and reduced blood flow
- deformation of the skeleton and the muscle apparatus
- general retardation of the embryo development
Reported statistics and error estimates:
- oedematous enlargement of the pericardial space in about 20 % of all treated embryos
- deformation of the skeleton and muscle apparatus in approximately one third of all treated embryos
- retardation of body development in about 20 % of all treated embryos

Any other information on results incl. tables

Table 1: Comparison of the toxicity of N-methylaniline to embryonal development of Danio rerio with the results for aniline and N,N-dimethylaniline:

Dimension

Concentration range

NOEL

LC50

95%-C.I.

LC100

Ratio-NOEL

Aniline

mmol

0.2 - 20

1

9.3

9.2-9.5

17

2.E-04

N-Methylaniline

umol

0.1 - 5

0.2

0.71

0.69-0.72

1

1

N,N-Dimethylaniline

umol

0.08 - 4

0.33

1.51

1.49-1.54

2

0.6

Hence, the fish-embryotoxicity of N-methylaniline is four orders of magnitude higher compared to aniline, whereas it is in the same range as N,N-dimethylaniline.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Remarks:
only similar to guideline
Conclusions:
In this study N-methylaniline was found highly toxic to Zebrafish embryo, with a NOEC of 0.2 µmol/L or 21 ug/L and a LC50 of 0.71 µmol/L or 76 ug/L.
Executive summary:

Zebra-fish embryos exposed for 96 h were more sensitive to methylanilines than to Nmethylamine, N,N-dimethylamine, 2-aminoethanol, and isopropylamine. N-methylanilie, like aniline and N,N-dimethylaniline, produced malformations in the fish embryos with lesions mainly of the heart or skeleton. Besides mortality, effects observed were: a general retardation of the embryo development and oedematous enlargement of the pericardial space with retardation of heart development and reduced blood flow. In addition, exposure to N-methylaniline caused a deformation of the skeleton and the muscle apparatus. The underlying cause of death could not be determined explicitly although an obvious association between malformations and death existed. The NOEL for N-methylaniline was 0.2 µmol/L, while the NOEL for aniline was 1 mmol/L. In conclusion: The embryotoxicity of aniline is enhanced considerably by its alkylation to N-methylaniline or N,N-dimethylaniline. However, NH2-substitution of alkyl compounds leads to agents with less embryotoxicity.