Isodecyl 3,5,5-trimethylhexanoate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April 2017 - 20 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

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:

yes

Details on sampling:

- Media Preparation Work:
After the range-finder test was conducted without chemical analysis a media trial was conducted to assess test substance solubility and stability in the test system. Media were prepared in duplicate, using a WAF preparation method. New media samples were taken at 0 hours, media were then stored in test conditions for ca 48 hours. Old media samples were then taken to assess stability.

- Chemical Analysis:
Chemical analysis was performed during the definitive test, however, analysis showed the batch did not meet acceptance criteria. Therefore, results could not be reported. Further media work showed there was insufficient sensitivity in the method, it was considered that the LOQ could not be lowered any further. The issues that led to this decision are summarised in the test substance properties and related issues section of the report.

Vehicle:

no

Details on test solutions:

- Preliminary Solubility Trial:
Based on the low solubility of the compound, which was stated as “insoluble in water” on the MSDS (material safety data sheet) it was considered necessary to conduct a trial to see what preparation method would be most appropriate. A saturated solution preparation was compared to a WAF (water accommodated faction) preparation. Both preparations were considered to be suitable for the compound, however, a WAF preparation was considered most suitable (OECD 23), even though the compound was a single component compound, but have multiple isomeric forms in the media.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name - Daphnia magna
- Source - MicroBio Tests Inc., Belgium.
- Age of parental stock (mean and range, SD):
- Feeding during test - none

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES
Culturing of Daphnia magna
Juvenile Daphnia magna (Straus), less than 24 hours old, were taken from healthy parental laboratory cultures, held and maintained at the test facility. The culture was originally obtained from Microbiotest, Belgium.

The culture was originally obtained from MicroBio Tests Inc., Belgium. The Daphnia magna are cultured in 1000 mL glass beakers containing 1000 mL of Elendt M4 medium. Each vessel and its contents are referred to as a 'culture'. New cultures are initiated with juvenile Daphnia magna (less than 24 hours old), at a density of 10-20 daphnids per vessel. The cultures are fed daily with a concentrated suspension of Chlorella vulgaris prepared in accordance with standard operating procedures.

The water in each culture was renewed or partially renewed at least once a week. Juveniles were removed when present in cultures using a sieve. Cultures were maintained up to a maximum of 42 days. Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before a test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use. All juveniles used to initiate a test were less than 24 hours old.

All cultures, prior to and during toxicity tests, are maintained under fluorescent lighting on a 16-hour light:8-hour dark photoperiod.

Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Not reported.

Test temperature:

Temperature Range (°C) 19.4 – 20.4

pH:

pH Range - 7.46-7.99

Dissolved oxygen:

Dissolved oxygen Range (Dissolved oxygen % ASV (mg/L)) - 98 (9.6) - 105 (10.02)

Salinity:

Not applicable

Conductivity:

Not reported.

Nominal and measured concentrations:

Range-finding test: 1.0, 10, and 100 mg/l loading rate
Definitive test: 100 mg/l loading rate

Details on test conditions:

- Range-finding test:
The range-finding test was conducted at nominal test substance concentrations of 1.0, 10 and 100 mg/L LR WAF under static test conditions. A control group was also included. Duplicate test vessels were prepared for the control and each test concentration. Based on nominal concentrations, the results of the range-finding test suggested that the compound was not toxic to daphnia. Five juvenile daphnia (<24 hours old) were added to each test vessel.

- Definitive Test:
Based on the results of the range-finding test, for which the key results only have been reported, the definitive limit test was conducted at a single test concentration of 100 mg/L LR WAF. The test was conducted with a semi-static test system design. Media was prepared at 0 and 24 hours.
The test vessels were 100 mL glass beakers (filled to approximately 50mL). Four replicate test vessels were prepared for the control and 100 mg/L LR WAF test concentration.
At the start of the test, an amount of test substance (100.09 mg) was added to 1000 mL of Elendt M4 medium. The preparation was stirred for ca 24 hours (with a vortex no deeper than ca 1 cm, 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 Elendt M4 medium only to the control vessels. At 24 hours media were prepared using the same method, 99.97 mg of test substance was weighed for use at 24 hours. Daphnia were then transferred to the new media prepared at 24 hours.
Five juvenile Daphnia magna, less than 24 hours old, were added to each test and control vessel, using a wide bore glass pipette to avoid damaging the animals during transfer. The Daphnia magna were not fed during the test.
After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility.
The observations differentiated between mobile and immobile daphnids. An individual was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation. In addition, Daphnia magna submerged in the body of the test media and those that were held at the surface of the test media were also recorded.

- Water Quality and Environmental Conditions:
The test was conducted with a 16-hour light: 8-hour dark photoperiod.
The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures were determined in freshly prepared test media at the start of the test and at media renewal at 24 hours and in the old media at 24 and 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the test area.
The freshly prepared media water quality measurements were performed on media sampled directly from preparation flasks. The old media water quality measurements were conducted using pooled replicate samples of old test media.

- Mathematical Rounding Statement:
Throughout the results, numerical data may have been rounded for presentation purposes. Therefore, manual recalculation of the data may result in slightly different values to those shown.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, Batch No. 13D290030, Purity: 100%, expiry date: 31 Jan 2018

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: loading rate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: loading rate

Details on results:

- Range-finding Test:
The results of the range-finding test are summarised below:
Chemical analysis was not conducted during the range-finding test.
The results of the range-finding test suggested that toxicity would not be observed at the highest concentration of 100 mg/L LR WAF.

- Test Substance Properties and Related Issues:
Chemistry analysis results did not meet acceptance during the definitive test. The reasons for this, and the decision to not report analytical results are discussed below. The test substance Isodecyl 3,5,5-trimethylhexanoate, is a moderately volatile compound with limited water solubility. As such, it presents as a good candidate for analysis by gas chromatography/mass spectrometry (GC/MS). This technique relies on initial separation on a chromatography column, mainly by the mechanism of differences in boiling point/volatility. Further separation and selectivity is provided by mass analysis, which also provides quantitative information, via the peak response for the analyte. Normally, GC/MS is considered to be a highly selective technique. However, many of the ions produced by the test substance are quite generic and might reasonably be expected to be produced by endogenous materials in the samples (e.g. lipids, fatty alcohols etc.).

Although the test substance, Isodecyl 3,5,5-trimethylhexanoate is a single chemical species, the isodecyl moiety within the molecule consists of a series of isomeric homologues. As each isomeric homologue has a slightly different boiling point, when the test article is analysed by GC/MS, a series of partially separated peaks, eluting as a broad “hump” is observed. This transforms the total peak response into a low, wide peak, with less well defined start and end points. This is at odds with the normal expectation in chromatographic techniques of measuring a single, sharp peak.

This has several impacts. First, by reducing the effective peak height there is a worsening of the signal to noise ratio, which is used to establish the limit of quantification (LOQ) of the analytical method. Therefore, the absolute sensitivity of the method is reduced.

Second, the start and end points of the peak are less well defined, which reduces both the accuracy and the precision of the measurement of peak areas (and hence the concentration).

Third, since the peak elutes across a wider time window than a sharp, narrow peak, it is much more difficult to say with certainty that there are no interferences extracted from the matrix lying beneath the analyte peak. Interferences producing the same ions s the test substance would not be excluded from the peak response, which will reduce the accuracy of the measurement.

Finally, Isodecyl 3,5,5-trimethylhexanoate was found to be more volatile than expected during concentration of the sample extract (performed to improve the LOQ). Although steps were taken to control and reduce these loses, it may have further contributed to the difficulties with the methodology.

Smithers Viscient Study Number 3201857 found the solubility of the compound to be less than the LOQ, the solubility was therefore considered to be < 0.01 mg/L.

An analytical method was developed and validated for use with the compound, however, batches ran throughout the duration of the study showed several issues. Achieved solubility were calculated as below the LOQ, meaning results could not be reported, other batches did not meet method criteria and therefore could also not be reported. The decision was made alongside the client to base results on nominal loading rate concentrations. It was considered that solubility of the compound was below the LOQ.

Media trial results are summarised below:

Results at 0 hours were below LOQ, results at 48 hours showed no concentration. Therefore, the Definitive phase was run semi-static.

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

- Toxicity to Daphnia magna:
The numbers of immobilised Daphnia magna at 24 and 48 hours, following exposure are presented in Table 2 and Table 3. The percentage immobility data is summarised below.

No significant toxicity (>10%) was observed during the test. It is therefore considered that the results showed the test substance was not toxic to the test organisms at 100 mg/L LR WAF or at the limit of solubility in the media.

- Validity Criteria:
The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Results with reference substance (positive control):

The results with reference substance are valid as the following criteria were met: The maximum permissible control or solvent control immobility is 10% at the end of the study; and the dissolved oxygen concentration must be maintained at ≥ 3 mg/L at the end of the exposure.
Treatment rates used were : Control, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. These were preapred by adding an amount of reference substance (ca 3.2 mg) was dissolved in 1000 mL of Elendt M4 Medium to give a 3.2mg/L top concentration, the remaining test mediums were prepared by serial dilution from the top
concentration down.
The study was a static regime, with test media preparation at 0 hours only. Temperature maintained between 18 and 22°C. pH between 6 and 9, oxygen was maintained > 3mg/L, 16:8
hours light:dark photoperiod. The Daphnia magna were observed after 24 and 48 hours exposure and the number immobilised in each vessel recorded.
The 48-hour EC50 value and NOEC were determined to be 0.626 and 0.32 mg/L, respectively. This 24 hour EC50 result is within the expected range stated in the OECD 202 testing guideline (0.6 – 2.1 mg/L).

Range-finding test results:

Nominal concentration (mg/l LR WAF)	Cumulative immobilised Daphnia magna*
	24 hours		48 hours		% Immobility
	R1	R2	R1	R2
Control	0	0	0	0	0
1	0	0	0	0	0
10	1	0	1	0	10
100	0	0	0	0	0

* Initial population of Daphnia magna: five per replicate

R Replicate

Media test concentration trial:

Nominal concentration (mg/l LR WAF)	Measured Concentrations (mg/l)
	9 hours	48 hours
100 (a)	(<LOQ)	NF
100 (b)	(<LOQ)	NF

LOQ 0.01 mg/l

NF None found

Toxicity to Daphnia magna:

Nominal concentration (mg/l LR WAF)	Cumulative immobilised Daphnia magna Iintial population :5 Daphnia per replicate
	24 hours					48 hours
	R1	R2	R3	R4	% immobility	R1	R2	R3	R4	% immobility
Control	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour acute toxicity of Isodecyl 3,5,5-trimethylhexanoate to the freshwater planktonic crustacean, Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).
The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.
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 48-hour acute toxicity of Isodecyl 3,5,5-trimethylhexanoate against the mobility of the freshwater planktonic

crustacean, Daphnia magna. The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).

Based on the results of a range-finding test, for which the key results only have been reported, a definitive limit test was conducted at a single concertation of 100 mg/L LR

WAF (loading rate water accommodated fraction). A control group was also included. The test was conducted under semi-static test conditions with media preparation at 0

and 24 hours. At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The

Daphnia magna were not fed during the test. Analytical chemistry was performed during the definitive phase, however, due to the solubility of the compound being below the LOQ (limit of quantification) of the analytical method, which was 0.01 mg/L, therefore, values could not be reported as they were not reliable. 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.

The EC50 values, the corresponding NOEC and LOEC values are presented in the following table.

Endpoint	Nominal concentration (mg/l)		Statistical Test
	24 hour	48 hour
EC50	>100 mg/l	>100 mg/l	Derived Empirically
NOEC	100 mg/l	100 mg/l
LOEC	>100 mg/l	>100 mg/l

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

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Description of key information

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

Additional information

The results indicated that there was no toxicity at the limit of solubility of the test substance. The NOEC is 100 mg/L