1,3-Cyclohexanedimethanamine, N1,N3-bis(2-methylpropylidene)-

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:

2010-11-15 to 2011-10-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study, in accordance to OECD, EU guidelines; 1st Experiment: Limit test, 2nd Experiment: Full test

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling preparation:
For the analysis of the test item concentration, samples from the test media of the test concentrations and the control were taken at the start and at the end of both experiments (Experiment 1 was terminated on Day 3, approximately 72 hours after the treatment). Representative samples in duplicate series were taken. One series were sent for analysis, and were analyzed immediately after sampling.
The samples at concentration level 100 mg/L were diluted twofold with water: The samples at concentration levels 6.25 - 12.5 - 25 and 50 mg/L and the control samples were measured directly.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Formulation: In Experiment 1 test solution at concentration of 100 mg/L was prepared in the dilution water (the same aquarium water that is used during adaptation of the fish) directly without use of any solubilising agent in a final volume of 5000 mL. Appropriate amount of the test item was weighed, blended with the dilution water, thereafter sonicated for 30 minutes in ultrasonic bath (under cooling). The resulted solution was centrifuged at 4000 rpm for 10 min. (20 °C) and filtrated using a 0.45 μm filter (VWR Vacuum Filtration System). pH value of treatment solution was not adjusted.
In Experiment 2 stock solution at concentration of 100 mg/L (nominal) was prepared in the dilution water (the same aquarium water that is used during adaptation of the fish) directly without use of any solubilising agent in a final volume of 10 L. Appropriate amount of the test item was weighed, blended with the dilution water, thereafter sonicated for 30 minutes in ultrasonic bath (under cooling). The resulted solution was centrifuged at 4000 rpm for 10 min. (20 °C) and filtrated using a 0.45 μm filter (VWR Vacuum Filtration System). pH value of the stock solution was not adjusted. Further test concentrations were diluted from the stock solution with the dilution water.

- Untreated Control: The dilution water (without addition of the test item) was used as a test solution.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Danio rerio
- Source: Fish Laboratory of TOXI-COOP Zrt. (8237 Tihany, Klebelsberg K. u. 3.).
- Length at study initiation (length definition, mean, range and SD): 2 ± 1 cm in both experiments
- The fish were not fed during the test.

ACCLIMATION
Since fish used in the test were bred in the laboratory, no acclimatization period was necessary prior to start of the test.
-Adaption: On Day (-8) the test fish were adapted to freshly prepared test water (i.e. dilution water; reconstituted (ISO) water). In both experiments the test fish were adapted to freshly prepared test water (i.e. dilution water; reconstituted (ISO) water) and were monitored for mortality (from Day (-11) and from Day (-7) in Experiment 1 and Experiment 2, respectively).
- Type and amount of food: During holding, until one day before the test start, the fish were fed with appropriate, commercial diet for fish.
- Health during adaption: No mortality was observed during the adaptation period.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Experiment 1 and 2:
246.5 and 231.4 mg CaCO3 per litre

Test temperature:

Experiment 1:
23.3 – 24.0 °C in the control
23.4 – 24.1 °C in the 100 mg/L treatment group

Experiment 2:
23.0 – 23.5 °C in the control
22.6 – 23.6 °C in the test item treated groups

pH:

Experiment 1:
7.38 – 7.57 in the control
7.79 – 9.25 in the 100 mg/L treatment group

Experiment 2:
7.32 – 7.40 in the control
7.19 – 8.92 in the test item treated groups

Dissolved oxygen:

Experiment 1:
89.1 –97.8 % of ASV in the control
69.3– 93.5 % of ASV in the 100 mg/L treatment group

Experiment 2:
88.3 –95.1 % of ASV in the control
68.6– 96.0 % of ASV in the 100 mg/L treatment group

ASV = Air Saturation Value

Nominal and measured concentrations:

Experiment 1 (Limit test):
Nominal: 100 mg/L

Experiment 2 (Full test):
Nominal: 6.25, 12.5, 25, 50, 100 mg/L
Mean measured concentration (with 95 % confidence interval): 6.48 ± 0.11, 11.8 ± 0.11, 23.6 ± 0.35, 47.0 ± 1.76, 92.9 ± 0.81

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquariums with 5 litre test liquid.
- No. of organisms per vessel: 7 animals at each group (no parallels were used) in each experiment
- Aeration: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light: 8 h dark

EFFECT PARAMETERS MEASURED:
NOEC: No Observed Effect Concentration: the highest test concentration at which no significant effect on the test animals is observed.
LOEC: Lowest Observed Effect Concentration: the lowest test concentration at which a significant effect on the test animals is observed.
LC 50: The calculated concentration of the test item which kills 50 % of the test fish.

TEST CONCENTRATIONS
- Range finding study: In the non-GLP preliminary test the test item concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 mg/L (nominal) were examined. The number of test animals in this preliminary test in every concentration levels and in the control was 2. Test solutions were not aerated during the test. Although pH value of the test solution at concentrations of 100 % and 50 % differed from the control by more than 1 unit (1.71 and 1.33, respectively) at the start of the test, no adverse effect caused by the pH change was observed in the preliminary test. No mortality or any other symptoms were observed during the preliminary test.
- Results used to determine the conditions for the definitive study: Based on the results of this range-finding test, a limit test (Experiment 1) was selected to be performed according to the relevant guideline (OECD 203). One control and the concentration of 100 mg/L (nominal) of the test item were tested. Since mortality was observed during this limit test a subsequent full test (Experiment 2) was performed with a range of 5 different test item concentrations.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

27.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL: 18.73 - 41.23 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Reported statistics and error estimates:

The 96 hour LC50 and the 95 %-confidence limits were calculated by appropriate statistical test (Probit analysis by PC/SPSS+ Statistical Software). The LOEC, NOEC, LC0 and the LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Analytical results:

The test item Incorez 397 decomposes in aquatic formulation. The decomposition product of Incorez 397 was analysed in the test solutions by HPLC method with UV detection according to validated method. Since mortality was observed during Experiment 1 (limit test) a subsequent full test was performed.

In Experiment 2 five different concentrations arranged in a geometric series (differing by a constant factor of 2) and an untreated control group was tested. The nominal test concentrations of Incorez 397 were 6.25, 12.5, 25, 50 and 100 mg/L. Three replicate samples were taken from each test concentrations and from the control at the start (Day 0) and at the end (Day 4) of the experiment. The corresponding measured concentrations of the decomposition product of Incorez in the samples varied in the range from 93 % to 106 % in comparison to the nominal values.

As the performed analytical determinations confirmed that the test item concentrations based on the decomposition product remained within the range of ± 20 % of the nominal, the biological results are reported on the basis of the nominal concentrations of the test item.

 

Clinical symptoms:

In Experiment 1 the following symptoms were observed at the 100 mg/L treatment group: unbalanced swimming (2/7 fish), decreased activity (7/7 fish), dorsal position (1/7 fish), lateral position (2/7 fish), increased motility of operculum (7/7 fish), closed fins (4/7 fish) and abnormal localization in the aquarium (7/7 fish). No clinical symptoms were observed in the control. In Experiment 2 no clinical symptoms were observed in the control group and in the 6.25 mg/L treatment group. The following symptoms were observed at the concentration level of 100 mg/L: unbalanced swimming (3/7 fish), decreased activity (7/7 fish), dorsal position (1/7 fish), lateral position (1/7 fish), closed fins (7/7 fish) and abnormal localization in the aquarium (7/7 fish). At concentration of 25 mg/L unbalanced swimming (2/7 fish), decreased activity (7/7 fish), closed fins (7/7 fish) and abnormal localization in the aquarium (7/7 fish) was observed. The following symptoms were recorded at concentration level of 12.5 mg/L: unbalanced swimming (1/7 fish), convulsive swimming (1/7 fish), decreased activity (1/7 fish), closed fins (1/7 fish) and abnormal localization in the aquarium (1/7 fish).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this acute fish toxicity study the observed endpoints for the effect of Incorez 397 were the followings: 96h LC50 value: 27.79 mg test item/L; 96h NOEC value: 6.25 mg test item/L and 96h LOEC value: 12.5 mg test item/L.

Executive summary:

Acute toxicity of the test item was assessed with Acute Fish Toxicity Test on Zebrafish (Danio rerio) over an exposure period of 96 hours in a static system. The recorded effects were mortality and symptoms of intoxication of the fish. Based on the results of this range-finding test, a limit test (Experiment 1) was selected to be performed according to the relevant guideline (OECD 203). One control and the concentration of 100 mg/L (nominal) of the test item were tested. Since mortality was observed during this limit test a subsequent full test (Experiment 2) was performed with a range of 5 different test item concentrations. The nominal test concentrations of Incorez 397 were 6.25, 12.5, 25, 50 and 100 mg/L. All test groups (in both Experiments) comprised seven zebra fish and 5 L test solution. No replicates were fulfilled.

In Experiment 1 mortality was observed in the test item treated group (100 mg/L): 3 of 7 fish died approximately 24 hours after the treatment and 100 % (7/7 fish) mortality was observed at approximately 72 hours after the treatment. No mortality was observed in the control group. In Experiment 2 mortality was observed in the 100, 50, 25 and 12.5 mg/L treatment groups. In the 100 mg/L test item treated group 100 % mortality (7/7 fish) was observed approximately 24 hours after the treatment. In the 50 mg/L test group 2 of 7 fish died 24 hours after the treatment and all fish found to be died 96 hours after the treatment. In the 25 mg/L and in the 12.5 mg/L test groups only 1-1 fish died during the test (48 hours after the treatment). No mortality was observed in the 6.25 mg/L treatment group and in the control.

Under the conditions of this acute fish toxicity study the observed endpoints for the effect of Incorez 397 were the followings: 96h LC50 value: 27.79 mg test item/L; 96h NOEC value: 6.25 mg test item/L and 96h LOEC value: 12.5 mg test item/L.

Description of key information

Under the conditions of this acute fish toxicity study the observed endpoints for the effect of Incorez 397 were the following: 96h LC50 value: 27.79 mg test item/L; 96h NOEC value: 6.25 mg test item/L and 96h LOEC value: 12.5 mg test item/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

27.79 mg/L

Additional information

Acute toxicity of the test item was assessed with Acute Fish Toxicity Test on Zebrafish (Danio rerio) over an exposure period of 96 hours in a static system. The recorded effects were mortality and symptoms of intoxication of the fish. Based on the results of this range-finding test, a limit test (Experiment 1) was selected to be performed according to the relevant guideline (OECD 203). One control and the concentration of 100 mg/L (nominal) of the test item were tested. Since mortality was observed during this limit test a subsequent full test (Experiment 2) was performed with a range of 5 different test item concentrations. The nominal test concentrations of Incorez 397 were 6.25, 12.5, 25, 50 and 100 mg/L. All test groups (in both Experiments) comprised seven zebra fish and 5 L test solution. No replicates were fulfilled.

In Experiment 1 mortality was observed in the test item treated group (100 mg/L): 3 of 7 fish died approximately 24 hours after the treatment and 100 % (7/7 fish) mortality was observed at approximately 72 hours after the treatment. No mortality was observed in the control group. In Experiment 2 mortality was observed in the 100, 50, 25 and 12.5 mg/L treatment groups. In the 100 mg/L test item treated group 100 % mortality (7/7 fish) was observed approximately 24 hours after the treatment. In the 50 mg/L test group 2 of 7 fish died 24 hours after the treatment and all fish found to be died 96 hours after the treatment. In the 25 mg/L and in the 12.5 mg/L test groups only 1 fish died during the test (48 hours after the treatment). No mortality was observed in the 6.25 mg/L treatment group and in the control.

Under the conditions of this acute fish toxicity study the observed endpoints for the effect of Incorez 397 were the followings: 96h LC50 value: 27.79 mg test item/L; 96h NOEC value: 6.25 mg test item/L and 96h LOEC value: 12.5 mg test item/L.