Registration Dossier

Administrative data

Endpoint:
long-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From April to July 2013
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)
Qualifier:
according to
Guideline:
other: GB/T 21808-2008 "Chemicals-Fish, prolonged toxicity: 14-day study test".
Qualifier:
according to
Guideline:
other: OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OEeD Series on Testing and Assessment, No. 23. Organisation for Economic Development and Cooperation, Paris, France. September 2000.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: white solid
Details on test material:
- Name of test material (as cited in study report): XP-7866
- Physical state: solid
- Analytical purity: 99.5 wt%
- Lot/batch No.: 10188-1JM
- Expiration date of the lot/batch: Feb 28, 2015
- Storage condition of test material: room temperature, 15-25 °C

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Three samples (each with 5ml) werc collected from test vessel and analyzed in the new media on 1 d, 2 d, 8 d, 9 d and 14 d, and in the expired media on 2 d, 8 d, 9 d, 14 d and 15 d. One sample was taken for analysis in the solvent and blank control group on each occasion. The samples taken on 1d, 8 d and 14 d were stored in refrigerator (2-8 °C) and analyzed with the samples taken on 2d, 9 d and 15 d.
- PRETREATMENT: 5 ml of sample was taken and equivalent volume methanol was added. The solutions were mixed thoroughly. About 1.5ml sample was used for HPLC analysis after filtering with a 0.22 µm membrane.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: negative and solvant control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethyl formamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): a stock solution with concentration of 5mg/ml of the test item in dimethyl formamide were prepared. Stored at 2-8°C. Then, 1 ml was taken from the stock solution into the glass jar of 10 L of test water every day.After fully stirring with magnetic force (with speed of ca. 400-450 rpm, time of ca. 2-4 hours), the test solution was transferred into a glass tank and fish were
exposed.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Batch: 20120613
- Feeding: daily (the quantity of food being kept constant)
- Loading: Less than 1.0 gfish/liter water

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
14 d

Test conditions

Hardness:
Measured before the test
Test temperature:
23 +/- 2°C
Dissolved oxygen:
Not less than 60% of ASV during the test period
Nominal and measured concentrations:
- 0.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: In this study, 10 L glass jars (diameter:20 cm, high:40 cm) and 10L glass tanks(30 cmx20cm x30 cm) were used.
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static
- Renewal rate of test solution (frequency/flow rate): renewal of the test solution every 24 h
- No. of organisms per vessel: 10 organisms in the test and control vessel
- No. of vessels per concentration (replicates): not reported
- No. of vessels per control (replicates): not reported
- No. of vessels per vehicle control (replicates): not reported
- Biomass loading rate: 1 g fish/liter water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The water used for the aquatic test was demineralized water treated with special softening techniques. Before usage, the water was aerated for at least 24 hours in order to achieve dissolved oxygen saturation.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 hours photoperiod daily
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
* Total length and weight measured at the start and at the end of the study
* observed daily for mortality and toxic signs
Mortality detremination: Lethal effects are defmed as follows: a fish is presumed to be dead if no respiratory movement and no reaction to a slight mechanical stimulus can be detected.

TEST CONCENTRATIONS:
The determination of the test concentration was based on the limit test acute toxicity. For details, refer to endpoint 6.1.1 Short-term toxicity to fish, report N° G1223J0070. - Test conc.: 0.5 mg/L
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
adult mortality
Details on results:
- Toxic Signs
During the 14-day exposure period, the fish treated with the test item showed no visible toxic signs and there were no deaths at the limited concentration of 0.5 mg/L (measured as 0.28 mglL).
- Body Weight and Body Length
The result of determination of body weight and body length showed that no significant change was found in the controls and the test group.
* at the start of the experiment:
- Control:
Body length: 2.8+/-0.1 cm (mean +/- SD)
Body weight: 0.4182 +/- 0.0293 (mean +/- SD)
- Solvent control:
Body length: 2.7+/-0.1 cm (mean +/- SD)
Body weight: 0.3960 +/- 0.0491 (mean +/- SD)
- Test concentration (0.5 mg/L - nominal conc.)
Body length: 2.7+/-0.1 cm (mean +/- SD)
Body weight: 0.4334 +/- 0.0287 (mean +/- SD)
* at the end of the test:
- Control:
Body length: 2.8+/-0.1 cm (mean +/- SD)
Body weight: 0.4384 +/- 0.0624 (mean +/- SD)
- Solvent control:
Body length: 2.8+/-0.1 cm (mean +/- SD)
Body weight: 0.4017 +/- 0.0443 (mean +/- SD)
- Test concentration (0.5 mg/L - nominal conc.)
Body length: 2.8+/-0.1 cm (mean +/- SD)
Body weight: 0.4333 +/- 0.0388 (mean +/- SD)
Results with reference substance (positive control):
No positive control
Reported statistics and error estimates:
No statistical method was used while the test was a limit test.

Any other information on results incl. tables

Additional information on the analytical analyses:

* Stability of HPLC system and Comparison of Stock Solutions

The results of HPLC stability test showed that the HPLC system was relatively stable (The relative standard deviation (RSD) for peak area was from 0.12% to 0.61 %, and the retention time was from 0.04% to 0.46%, in the allowed range of <=3%).

The result of comparing the diluted standard solutions of the two stock solutions showed that the Relative Deviations (RD) of the two solutions were 1.2%, 12.9% and 6.2%, 2/3 were out of the rangc of ±5%. While the results of fortified samples in the same analysis batch showed that all the recoveries samples met the allowed ranges of 80%-120% demanded by the study plan, which means

the deviation did not affect the quality or integrity of the study.

* Verification of Test Concentrations from Definitive Test

The concentrations were calculated from the calibration line. The results showed that the mean measured concentration in the freshly prepared solutions was 0.30 mg/L, with mean concentration of 0.25 mg/L measured in the expired solutions, and the over mean concentration in the test samples, calculated using geometric means, was 0.28 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Following the OECD guidance n°204 under GLP conditions, the result showed the no observed effect concentration (NOEC) of the test item to zebra fish was 0.28 mg/L when exposed during 14 days.
Executive summary:

XP-7866 fish, prolonged toxicity test: A 14-day study with zebra fish (Dania rerio) was conducted.

Based on the acute test (refer to endpoint 6.1.1, report n°G1223J0070), a 'limit test' was performed with 0.5 mg/L in the definitive test. A concurrent control and solvent control were performed in parallel. Ten fish were used in the test and the controls groups. The

definitive test was performed with a semi-static water renewal system and the water used was demineralized water. Dissolved oxygen and pH values were determined at 1st, 8th, and 14th for the fresh and 2nd, 9th , and 15th for the expired solutions during the 14-day exposure period.

The concentration of test item in the test media was monitored by a reverse-phase high performance liquid chromatography (HPLC) method, using an external standard at 1st, 2nd, 8th, 9th, and 14th for the fresh and 2nd, 9th, and 15th for the expired solutions during the 14-day exposure period. The results showed that the geometric mean concentration of test item was measured as 0.28mg/L.

During the 14-day exposure period, no toxic signs and deaths were observed for the exposed fish either in the controls or the test group.

The result of the 14 days prolonged toxicity test showed the no observed effect concentration (NOEC) of the test item to zebra fish was 0.28 mglL.