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Ecotoxicological information

Long-term toxicity to fish

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Administrative data

Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
other: experimental study on the main metabolite
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Relative embrotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies
Author:
Hermsen SAB, van den Brandhof EJ, van der Ven LTM, Piersma AH
Year:
2011
Bibliographic source:
Toxicology in vitro, 745-53

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Zebrafish embryo toxicity test
Principles of method if other than guideline:
Fertilised zebrafish embryos are allowed to develop for 72 hours then subject to a full morphological evaluation by microscope. The test is designed as a screening study for mammalian developmental toxicity potential and in this case was intended to assess the toxicity potential of the main metabolite of 2-(2-methoxyethoxy)ethanol, i.e. 2-(2-methoxyethoxy)acetic acid (MEAA)
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
impurity
Specific details on test material used for the study:
Supplied by Sigma Aldrich

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Source: Originally Ruinemans Aquarium BV, Montfort, Netherlands but bred in test facility for 3 years.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Three days before spawning, femailes separated and fed only thawed Artemia. Males and females paired in spawning boxes (ratio 2:2). Spawning triggered by turning light off and usually completed in 30 mins.
- Method of collection of fertilised eggs: Collected 30 mins after spawning
- Subsequent handling of eggs, embryos and larvae: Rinsed.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
Incubated at 26.5 +/- 0.5C
pH:
7.4 - 8.4
Nominal and measured concentrations:
7 concentrations over the range 0.01 to 10mM. Not specified but shown on graphically presented results.
Details on test conditions:
TEST SYSTEM
- Test vessel: 24 well plants
- Material, size, headspace, fill volume: 2ml per well
- Renewal rate of test solution (frequency/flow rate):
- No. of fertilized eggs/embryos per well: 1
- No. of wells per concentration (replicates): 10
- No. of wells per control (replicates): 4
- Whole experiment was repeated in triplicate

OTHER TEST CONDITIONS
- adjustment of pH: Yes
- Photoperiod: 14 light, 10 dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Morphological evaluation of the embryos was performed at 72 h post fertilization (hpf) using amicroscope. A general morphology scoring system was used adapted from the system used for whole embryo culture using a semi-quantitative assessment of specific developmental endpoints. An experimental embryo is compared to the reference embryo in the scoring matrix and receives points for each developmental hallmark dependent on its stage of development. All deviations, for instance incomplete detachment of the tail, will result in a lower point score which corresponds to a certain extent of developmental retardation. Malformations and other teratogenic effects were separately recorded as present or absent. The list of teratogenic effects scored included: Pericardial edema, Yolk sac edema Eye edema. Malformation of the head, Malformation of sacculi/otoliths, Malformation of tail, Malformation of heart, Modified chorda structure, Scoliosis Rachischisis, Yolk deformation.
Reference substance (positive control):
yes
Remarks:
methoxyacetic acid, ethoxyacetic acid

Results and discussion

Effect concentrations
Remarks on result:
other: No adverse effects seen
Details on results:
No adveres effects were seen up to the maximum tested dose of 10mM.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- No effects were also seen with the glycol ether methoxyethanol. In contrast, the positiive control methoxyacetic acid produced a BMC5 for general morphology changes of 2.7mM (confidence interval 1.9-3.6) and for teratogenicity of 4.6 (2.5-5.7) whilst for ethoxyacetic acid, the BMC5 for general morphology changes was 3.6mM (confidence interval 2.6-3.7) and for teratogenicity of 2.9 (2.2-3.5). All of these values overlap and can be considered to be insignificantly different from one another.

Any other information on results incl. tables

It should be noted that the zebra fish model does not have metabolic capacity, which explains why methoxyethanol produced no effects. It is well established that the proximate toxicant is the acid metabolite and not the parent glycol ether. This study established that the acid metabolite of DEGME does not appear to share the same developmental toxicity characteristics as MAA.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
2-methoxyethoxy acetic acid, the main metabolite of 2-(2-methoxyethoxy)acetic acid does not appear to have teratogenic properties when evaluated using the zebrafish embryotoxicity test.
Executive summary:

2-(2-methoxyethoxy)acetic acid, the main metabolite of 2-(2-methoxyethoxy)ethanol was evaluated using the zebrafish embryotoxicity test (ZET) usingt a modified protocol desinge as fast and simple method to study chemical toxicity after exposure of the complete vertebrate embryo during embryogenesis in ovo. The protocol included a novel quantitative evaluation method to assess the development of the zebrafish embryo based on specific endpoints in time, the general morphology score (GMS) system , and a teratogenic evaluation using separate scoring system based on that used for whole embryo cultures. Methoxyethoxy acetic acid did not show any evidence of developmental toxicity up to the maximum tested dose of 10mM. In contrast, the know teratogencic substances

methoxyacetic acid and ethoxyacetic acid caused both growth retardation and malformations. A comparison of the results obtained with this substance and the others tested with the available mammalian developmental toxicity data showed the potency ranking of the compounds within their class in the ZET was comparable to their in vivo ranking.