Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-04 to 2012-05-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A saturated solution was tested
- Sampling method: Analytical evaluation of the saturated solution of Vestanat B 1370 (solvent free) and the control was carried out via LC-MS/MS from freshly prepared media after 0 h and 72 h and from corresponding 24 h aged test media after 24 h and 96 h.

- Sample storage conditions before analysis: All stabilized samples were stored at 6 ± 2 °C until sample preparation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Two stock solutions of 3.9 mg/L (2 x 5 L) were prepared once with dilution water three days prior to start of the exposure.

The test item dispersion was stirred for three days with a magnetic stirrer at room temperature. After a separation of the phases after 1 min of standing, the saturated solution was taken from the homogeneous phase and used for testing without further dilution.

Thereafter the stirring of the stock solutions was continued until day 3 of the study. For daily renewal of the test media of the limit concentration the above mentioned separation procedure was carried out (until last renewal of the test media on day 3).


- Eluate: Dilution water
- Differential loading: Saturated solution
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at DR.NOACK-LABORATORIEN from a single brood stock. (supplier: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, An der Scharlake 39, D-31135 Hildesheim)
- Length at study initiation (length definition, mean, range and SD): Average body length: 2.23 cm
- Weight at study initiation (mean and range, SD): Average body weight: 0.17 g
- Method of breeding:Holding was performed at the test facility at 23+/-2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test



ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 50 mg/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): daily renewal (after 0, 24, 48, 72 hours)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all vessels at the beginning of the test and every 24 h (from old and new media). Total hardness of the water was determined at the beginning out of the test control. During the test the water temperature was recorded continuously with a data logger (once per hour).


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural light
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1 (corresponding to approximately 7 - 750 Lux).


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: Yes
Cumulative Mortality [%] in the Preliminary Test (non GLP)
(n = 5)

Nominal test item concentration l
[mg/L] Test Duration [hours]
24 48 72 96
Saturated
solution 0 0 0 0
Control 0 0 0 0

Measured concentrations in the Preliminary Test

Sampling date 2012-02-28 0 h 2012-02-29 24 h 2012-02-29 48 h 2012-03-01 72 h
(Fresh medium) (Old medium) (Fresh medium) (Old medium)

VESTANAT B 1370 (solvent free)
Nominal
concentration
[mg/L] Measured concentration [µg/L]
Saturated solution 657 378 720 302

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

462 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 462 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test solution was clearly dissolved throughout exposure.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

The concentration leading to 0 and 100 % mortality (LC0 and LC100) after 96 h were determined directly from the test results. LC10,20,50-values have not to be calculated in a limit test.

Sublethal observations / clinical signs:

Observations in the Test Vessels

                        (n=7)

Geometric mean measured test item concentration [µg/L]	Effect*	Test Duration [h]
		24	48	72	96
462	(1)	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7

Cumulative Mortality [%] in the Test Vessels

 

Geometric mean measured test item concentration [µg/L]	Test Duration [h]
	24	48	72	96
462	0	0	0	0
Control	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

In this study Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked was found to cause no toxic effects to Zebrafish after 96 hours when tested with the saturated solution. The LC50 after 96 hours is > 462 µg/L. All effect levels are given based on the geometric mean measured concentration of the test item.

Executive summary:

The acute toxicity of the test item Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992). A semi-static limit test with daily renewal of the test media was performed with the saturated solution* of nominal 3.9 mg/L. The duration of the test was 96 h. Seven test organisms were exposed to the saturated solution and the control. Water quality parameters pH-value, temperature and oxygen-saturation measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits. The determination of the test item was carried out via LC-MS/MS from the saturated solution and the control from freshly prepared media after and 72 h and from 24 h aged test media after 24 h and 96 h.

The measured concentrations of Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked in freshly prepared media were 397 and 896 µg/L and in 24 h aged test media 227 and 563 µg/L, respectively. All effect levels are given based on the geometric mean measured concentrations of the test item.

LC-Values with 95 % Confidence Intervals

based on the geometric mean measured concentration of the test item [µg/L]

Test duration [h]	LC50	p = 95 %
24	> 462	n.a.
48	> 462	n.a.
72	> 462	n.a.
96	> 462	n.a.
LC100 =	> 462
Lowest test item concentration
with 100 % mortality after 96 h
LC0 =	462
Highest test item concentration
with 0 % mortality after 96 h

Description of key information

The acute toxicity of the test item Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992). A semi-static limit test with daily renewal of the test media was performed with the saturated solution* of nominal 3.9 mg/L. The duration of the test was 96 h. Seven test organisms were exposed to the saturated solution and the control. The determination of the test item was carried out via LC-MS/MS from the saturated solution and the control from freshly prepared media after and 72 h and from 24 h aged test media after 24 h and 96 h. The values were 397 and 896 µg/L (freshly prepared media) and 227 and 563 µg/L (in 24 h aged test media), respectively. All effect levels are given based on the geometric mean measured concentrations of the test item.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Remarks:

saturated solution

Effect concentration:

> 462 µg/L

Additional information

In this study Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked was found to cause no toxic effects to Zebrafish after 96 hours when tested with the saturated solution. The LC50 after 96 hours is > 462 µg/L. All effect levels are given based on the geometric mean measured concentration of the test item.