Isodecyl 3,5,5-trimethylhexanoate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April 2017 to 28 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 236 (Fish embryo acute toxicity (FET) test)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Substance Name: Isodecyl 3,5,5-trimethylhexanoate
CAS Number: 59231-35-5
Purity: 100%
Receipt Date: 01 March 2017
Storage on Receipt: Room Temperature (15 – 30°C)
Disposal: None hazardous
Hazard: None hazardous

Test substance details were supplied by the Sponsor. A Certificate of Analysis was not available for the compound; therefore, the client confirmed “the substance is a UVCB, a mixture of isomers and therefore assumed to be 100% pure”. Emails included in study data.

Analytical monitoring:

no

Remarks:

Analytical chemistry was not performed during the definitive test. As the solubility of the compound was below the LOQ (limit of quantification) of the analytical method (<0.01 mg/L).

Vehicle:

no

Details on test solutions:

- Dilution Water:
The dilution water used for conducting the tests was treated mains water that had been passed through activated carbon filters. The typical constituents of the water are presented in the report.

- Preparation of Test Media:
-Range finding test:
The range-finding tests were conducted at nominal concentrations of 0.10, 1.0, 10 and 100 mg/L LR WAF under static test conditions with media preparation at 0 hours only. Control and positive control groups were also included. Ten individual eggs were exposed for the control and each test group long with four internal controls. Based on nominal concentrations, the results of the range-finding test suggested that the 96-hour LC50 value would be greater than 100 mg/L LR WAF. A WAF preparation method was considered to be most appropriate for this compound as it was an Unknown or Variable Composition, Complex Reaction Products and Biological Material (UVCB).

- Definitive test:
in agreement with the Sponsor the definitive test was conducted at a nominal concentration of 100 mg/L LR WAF. At the start of the test, an amount of test substance (100.17 mg) was added to 1000 mL of Treated Mains Water. The preparation was stirred for ca 24 hours, media were then allowed to settle for ca 1 hour, final media were then syphoned from the mid-section (aqueous phase) of the vessel. A control treatment was prepared by adding Treated Mains Water only to the control vessels. At 24, 48 and 72 hours media were prepared using the same method, 100.13 mg of test substance was weighed for use at 24 hours, 100.05 mg for use at 48 hours, and 100.06 mg for use at 72 hours.
A positive control was also included using 3,4-dichloroaniline at a single concentration of 4.0 mg/L, this was prepared by adding the following weights of substance to 1000 mL of treated mains water, 4.00 mg for 0 hour preparation, 4.06 mg for 24 hour preparation and 4.02 mg for 48 hour preparation, however, the 48 hour preparation was discarded as all embryo in the positive control well plate we dead. This media was subsequently not prepared for the 72 hour media preparation.
The test was conducted using a semi-static test regime whereby the test solutions were replaced after each approximate 24-hour period.

- Appearance of Test Media:
The appearance, colour and behaviour of the test substance in the test media were recorded daily throughout the test. All test media throughout definitive test were observed as colourless solutions.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Test Organism
The Danio rerio (zebra fish) eggs used in this study were obtained from an in-house laboratory breeding system.
Mating and spawning was conducted in plastic mating tanks with a plastic mesh at the bottom through which eggs could fall and be collected. Males and females were separated using a plastic divider in the tank which was removed when the lights were on to initiate spawning/mating.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Ca: 24.1 mg/l

Test temperature:

25.2 °C to 26.2 °C

pH:

7.31 to 7.81

Dissolved oxygen:

95 (8.15) - 113 (9.75) %ASV (mg/l)

Salinity:

Not applicable

Conductivity:

218 µs/cm at 20 °C

Nominal and measured concentrations:

100 mg/l (loading rate)

Details on test conditions:

- Test Vessel Preparation:
The test vessels were 24-well microtiter plates. A single plate was used for the control, each test concentration and the positive control. Twenty wells on each plate were filled with 2 mL of the appropriate control or test media. The remaining four wells in each plate were filled with treated mains water to act as internal controls.

- Egg Addition and Observations:
Following collection, approximately 230 fertilised eggs were placed in crystallising dishes containing the appropriate control, test concentration or positive control concentration no later than 60 minutes post-fertilization and acclimatised to laboratory conditions. The eggs were less than one and a half hours old when added to the test vessels. Fertilized eggs were separated from non-fertilised eggs and transferred to the corresponding well plates containing the test solutions within 1.5 hours of fertilization.
At this point the eggs were at the start of 2-cell stage. A single fertilised egg was added to each well of the 24-well microtiter plates.
After ca 24, 48, 72 and 96 hours, observations were performed on the eggs for coagulation of the egg, absence of somite formation, non-detachment of the tail and lack of heart beat, where appropriate. The egg was considered to be dead if a positive response to any of these lethal endpoints was observed. The total number of dead eggs was recorded.

- Test Media Descriptions:
The test preparations were observed to be colourless solutions throughout the duration of the test.

- Water Quality and Environmental Conditions:
The test was conducted in a temperature and light controlled incubator (16 hour light:8 hour dark). At the start and end of the test and at each media renewal the pH, dissolved oxygen (% air saturation value and mg/L) and temperatures were carried out in the controls and the test concentration. The temperature was recorded continuously using a max/min thermometer. The water quality determinations taken during the definitive test are presented in the report. All values were considered to be within acceptable limits.

Reference substance (positive control):

yes

Remarks:

A positive control was also included using 3,4-dichloroaniline at a single concentration of 4.0 mg/L

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks on result:

other: No toxicity was observed at the solubility level of the compound in test media.

Details on results:

- Toxicity to Danio rerio embryos:
Embryo mortality recorded during the definitive test and hatching is presented in the report. The same parameters for the internal controls are also presented in the report.
A summary of the percentage survival after 96-hours during the definitive test is provided below:

Nominal concentration (mg/L LR WAF) 96-hour survival (%)
Control 90
Positive Control 0
Test substance (100) 95

LC50 values are presented in the table below.

Toxicity value (mg/L LR WAF)
Parameter 24 hours 48 hours 72 hours 96 hours
LC50* >100 >100 >100 >100
*95% confidence limits were not calculated
Results derived imperially, statistical analysis not performed aas no toxicity observed during test.

The results indicated that there was no toxicity at the limit of solubility of the test substance.

The results of the third 96-hour range-finding test are summarised in the table below:
Nominal concentration 96-hour survival (%)
(mg/L LR WAF)
Control 90
Positive Control 0
0.10 70
1.0 100
10 100
100 90

The above results indicated that the LC50 value would be greater than 100 mg/L LR WAF.

- Validity Criteria:
The following validity criteria were all achieved and therefore the test is considered valid:
The fertility rate was 94%, which was greater than the validity criterion of ≥70%.
The water temperature was maintained at 26 ± 1°C during the test. The temperature ranged from 25.0 to 26.4°C.
The overall survival in the control was 90%, which was equal to the validity criterion of ≥90%.
For the reference substance, 3,4-dichloroaniline, the observed mortality was 100% which was in excess of the minimum 30% validity criteria.
The hatching success in the control was 94% at the end of the test, which was greater than the validity criteria of ≥80%.
The dissolved oxygen concentration was maintained above 80% of the air saturation value (ASV).

Results with reference substance (positive control):

A positive control was also included using 3,4-dichloroaniline at a single concentration of 4.0 mg/L, this was prepared by adding the following weights of substance to 1000 mL of treated mains water, 4.00 mg for 0 hour preparation, 4.06 mg for 24 hour preparation and 4.02 mg for 48 hour preparation, however, the 48 hour preparation was discarded as all embryo in the positive control well plate we dead. This media was subsequently not prepared for the 72 hour media preparation.

Reported statistics and error estimates:

Results derived imperially, statistical analysis not performed as no toxicity observed during test.

Sublethal observations / clinical signs:

A summary of the percentage survival after 96-hours during the definitive test is provided below:

Nominal concentration (mg/L LR WAF)	96-hour survival (%)
Control	90
Positive Control	0
Test substance (100)	95

LC50 values are presented in the table below.

	Toxicity value (mg/L LR WAF)
Parameter	24 hours	48 hours	72 hours	96 hours
LC50*	>100	>100	>100	>100

*95% confidence limits were not calculated

Results derived imperially, statistical analysis not performed aas no toxicity observed during test.

Validity criteria fulfilled:

yes

Conclusions:

The effects of the test substance on Danio rerio embryos by comparison of lethal endpoints with the controls were determined over a 96-hour period. The study was conducted in accordance with the known requirements of OECD Chemicals Testing Guideline No. 236: Fish Embryo Acute Toxicity (FET) Test (adopted 26 July 2013). All validity criteria were met during the test. Based on nominal loading rate concentrations, the 48-hour EC50 value was determined to be >100 mg/L LR WAF. The corresponding No Observed Effect Concentration (NOEC) was considered to be 100 mg/L LR WAF. No toxicity was observed at the solubility level of the compound in test media.

Executive summary:

The objective of the study was to determine the effects of the test substance on Danio rerio embryos by comparison of lethal endpoints with the controls to identify LC50 values. The lethal endpoints assessed, where appropriate, were; coagulated eggs/mortality, lack of somites, non-detachment of the tail and lack of heart beat. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 236: Fish Embryo Acute Toxicity (FET) Test (adopted 26 July 2013).

The definitive limit test was conducted at a nominal concentration of 100 mg/L LRWAF (loading rate water accommodated fraction). The concentration was based on the results of three range-finding tests, which are not fully reported. A control group was also included.

Test vessels (24-well microtiter plates) were prepared containing 2 mL of the appropriate test or control medium. To each well, a single fertilised egg was introduced. The study was conducted using a semi-static test design with daily renewal of test media for a period of 96 hours.

Analytical chemistry was not performed during the definitive test. As the solubility of the compound was below the LOQ (limit of quantification) of the analytical method, which was 0.01 mg/L, therefore, results were based on nominal loading rate concentrations (mg/L LR WAF). Smithers study number 3201857 found the water solubility to be less than the LOQ of 0.01 mg/L. Therefore, it was considered that the water solubility level was <0.01 mg/L.

1LC50 values are presented in the following table:

	Toxicity value (mg/L LR WAF)
Parameter	24 hours	48 hours	72 hours	96 hours
LC50*	>100	>100	>100	>100

*95% confidence limits were not calculated

Results derived imperially, statistical analysis not performed aas no toxicity observed during test.

The results indicated that there was no toxicity at the limit of solubility of the test substance.

All established validity criteria were met, therefore, the test was considered valid.

Description of key information

The key study was conducted according to internationally recognised testing guidelines and with GLP certification.

Additional information

Based on nominal loading rate concentrations, the 48-hour EC50 value was determined to be >100 mg/L LR WAF. The corresponding No Observed Effect Concentration (NOEC) was considered to be 100 mg/L LR WAF. No toxicity was observed at the solubility level of the compound in test media.