1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives (upper limit: 41% w/w; typical concentration: 38% w/w)

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2014 - June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

yes

Remarks:

In Tier 2 of the study only two test temperatures (10 and 30°C) have been applied

Principles of method if other than guideline:

In Tier 2 of the study only two test temperatures (10 and 30°C) have been applied

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Calibration standards
For preparation of calibration standards, a stock solution of the test substance was made in acetonitrile.
Calibration standards in water were prepared in the concentration range 0.2 – 2.5 mg/L (with a
maximum of 10% acetonitrile in water). The HPLC system with the settings described in Table I was
used for analysis. The prepared stock solutions in acetonitrile and calibration standards were stored in
the refrigerator until use.

Aqueous samples
In the beginning of the study, samples of the buffer solutions were analyzed directly after sampling
using the HPLC settings as described in Table I. In a later stage of the study, 9 mL of the buffers
solutions were diluted with 1 mL of acetonitrile and analyzed using the HPLC settings as described in
Table I.

Buffers:

Sterile test buffer solutions of pH 4, 7 and 9 were prepared in glass bottles according to the description
in Annex 3 of OECD 111 and purged with nitrogen for at least 5 minutes.

Details on test conditions:

Screening experiment (Tier 1)
A stock solution with a test substance concentration of approximately 1 g/L was prepared in acetonitrile. An amount of stock solution was spiked to a 100 mL volumetric flask and filled up with the respective buffer solutions, resulting in an initial concentration of 2 mg/L. Subsequently about 10 mL of the spiked buffer solution was transferred to multiple sterile glass test vials. The vials were closed tightly and placed in athermostatically controlled water bath in the dark at a temperature of 50 ± 0.5°C. At the moment the test vials were placed in the water bath, the first sample was taken and analyzed using the analytical method described in Annex 3. Subsequent samples were taken on different time intervals and analyzed to determine the percentage of hydrolysis. Samples were analyzed directly after sampling in order to prevent further hydrolysis. To check the pH of the buffer solutions at the test temperature, a separate glass bottle for each pH value was filled with buffer solution, but without test substance, and placed in the thermostatically controlled water bath (at the test temperature). After equilibration with the temperature of the water bath, the pH of this bottle was checked.

Hydrolysis of unstable substances (Tier 2)
A stock solution with a test substance concentration of approx. 1 g/L was prepared in acetonitrile. An amount of stock solution was transferred to a volumetric flask of 100 mL and filled up with the buffer solution, resulting in an initial concentration of 2.22 mg/L. Subsequently about 10 mL of the spiked buffer solution was transferred to sterile glass test vials. The vials were closed tightly and placed in the dark in the thermostatically controlledwater bath at two different temperatures of respectively 10.0 ± 0.5°C and 30.0 ± 0.5°C. At the moment the test vials were placed in the water bath, thefirst sample was taken and analyzed using the analytical method described in Annex 3. Sampling times were chosen based on the available results. For each sampling time duplicate sample bottles were prepared and both sample bottles were analyzed separately. A separate glass vessel without test substance was placed in the water bath at the different test temperatures. After equilibration with the temperature of the water bath the pH was checked of this vessel. The temperature of the thermostatic water bath was checked at the start and end of the tests and at each sampling time.

Number of replicates:

Duplicates

Positive controls:

no

Negative controls:

no

Preliminary study:

percentages, were considered to be representative for the test substance (AkzoNobel, 2014). Results for
these two constituents are presented.
The screening hydrolysis study was performed separately for each pH value tested. Results of the
different pH values are displayed below. A summary of the validation parameters of the analytical
method is given in Table I. Detailed information on the analytical method is given in Annex 3.
pH 4
The pH value at the start of the test was measured to be 4.0. The temperature during the test varied
between 49.9 and 50.1°C. It was observed that more than 80% of the test substance was hydrolysed
after 5 days. Detailed results of the hydrolysis are displayed in Table II and Figure 1.
pH 7
The pH value at the start of the test was measured to be 7.1. The temperature during the test varied
between 49.9 and 50.1°C. It was observed that between 62 and 80% of the test substance was
hydrolysed after 48 hours whereas a decrease to 13 and 38% after 5 days was observed. Detailed
results of the hydrolysis are displayed in Table III and Figure 2.
pH 9
The pH value at the start of the test was measured to be 9.0. The temperature during the test varied
between 49.9 and 50.1°C. It was observed that more than 80% of the test substance was hydrolysed
after 48 hours. Detailed results of the hydrolysis are displayed in Table IV and Figure 3.
As more than 10% of hydrolysis after 5 days was observed in Tier 1 for all pH values, also Tier 2 had to
be performed for the three tested pH values.

Transformation products:

not measured

pH:

4

Temp.:

10 °C

DT50:

>= 20 - <= 63 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Constituent 2/Constituent 1

pH:

4

Temp.:

30 °C

DT50:

>= 10 - <= 13 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Constituent 2 / Constituent 1

pH:

7

Temp.:

10 °C

DT50:

>= 9 - <= 19 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Constituent 2 / Constituent 1

pH:

7

Temp.:

30 °C

DT50:

>= 44 - <= 70 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Constituent 2 / Constituent 1

pH:

9

Temp.:

10 °C

DT50:

>= 58 - <= 80 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Constituent 2 / Constituent 1

pH:

9

Temp.:

30 °C

DT50:

>= 31 - <= 45 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Constituent 2 / Constituent 1

Details on results:

In Tier 2 of the study initially three test temperatures were used: 10, 30 and 50°C. The tests at 50°C
however showed very inconsistent results. This was most probably caused by the thermal instability of
the test substance. Therefore it was decided to confine Tier 2 of the study to 10 and 30°C, this is also
reflected in the second amendment to the study plan.
Results for the different pH values and test temperatures are described below. A summary of the
validation parameters of the analytical method is given in Table I. Detailed information on the analytical
method is given in Annex 3.
pH 4
Temperature 10°C
The temperature throughout the test was measured to be 10.0°C. The pH of the buffer solution was
checked at the start of the test and was measured to be 4.0. The results of both constituents showed
variable figures for hydrolysis. However after 30 days 20 – 24% of hydrolysis for constituent 1 and 52 –
72% of hydrolysis for constituent 2 was observed. The results were used to calculate the half-life (t0.5),
applying the Arrhenius relationship. The average half-lives were calculated to be 63 days for constituent 1
and 20 days for constituent 2.
Temperature 30°C
The temperature during the test varied between 29.8 and 30.0°C. The pH of the buffer solution was
checked at the start of the test and was measured to be 4.1. After 30 days about 82% of hydrolysis for
constituent 1 and about 88% of hydrolysis for constituent 2 was observed.The results were used to
calculate the halflife (t0.5), applying the Arrhenius relationship. The average half-lives were calculated
to be 13 days for constituent 1 and 10 days for constituent 2.

Using the determined half-lives at the different test temperatures interpolation curves for both
constituents were composed. Using the equations of these interpolation curves, the halflives
at a temperature of 25°C were interpolated to be 25 days for constituent 1 and 13 days for
constituent 2.

pH 7
Temperature 10°C
The temperature throughout the test was measured to be 10.0°C. The pH of the buffer solution was
checked at the start of the test and was measured to be 7.0. After 30 days 46 – 67% of hydrolysis for
constituent 1 and 81 – 89% of hydrolysis for constituent 2 was observed. The results were used to
calculate the halflife (t0.5), applying the Arrhenius relationship. The average half-lives were
calculated to be 19 days for constituent 1 and 9 days for constituent 2.
Temperature 30°C
The temperature during the test varied between 29.9 and 30.0°C. The pH of the buffer solution was
checked at the start of the test and was measured to be 7.0. After 32 days 20 – 22% of hydrolysis for
constituent 1 and 33 – 34% of hydrolysis for constituent 2 was observed. The results were used to
calculate the half-life (t0.5), applying the Arrhenius relationship. The average half-lives were
calculated to be 70 days for constituent 1 and 44 days for constituent 2.

Using the determined half-lives at the different test temperatures interpolation curves for both
constituents were composed. It is to be noted the calculated half-life for 30°C is higher
than for 10°C whereas it would be expected reverse. Using the equations of these interpolation curves,
the half-lives at a temperature of 25°C were interpolated to be 57 days for constituent 1 and 35 days for
constituent 2.

pH 9
Temperature 10°C
The temperature throughout the test was measured to be 10.0°C. The pH of the buffer solution was
checked at the start of the test and was measured to be 9.1. After 29 days 17 – 19% of hydrolysis for
constituent 1 and 25 – 26% of hydrolysis for constituent 2 was observed. The results were used to
calculate the half-life (t0.5), applying the Arrhenius relationship. The average half-lives were
calculated to be 80 days for constituent 1 and 58 days for constituent 2.
Temperature 30°C
The temperature during the test varied between 29.9 and 30.0°C. The pH of the buffer solution was
checked at the start of the test and was measured to be 9.0. After 30 days 32 – 56% of hydrolysis for
constituent 1 and 40 – 71% of hydrolysis for constituent 2 was observed. The results were used to
calculate the half-life (t0.5), applying the Arrhenius relationship. The average half-lives were
calculated to be 45 days for constituent 1 and 31 days for constituent 2.

Using the determined half-lives at the different test temperatures interpolation curves for both
constituents were composed. Using the equations of these interpolation curves, the halflives
at a temperature of 25°C were interpolated to be 54 days for constituent 1 and 38 days for
constituent 2.

pH 4
	Constituent
	1	2
Temp	Half-life	Half-life	Remark
°C	days	days
10	63	20	determined
30	13	10	determined
25	25	13	interpolated

pH 7
	Constituent
	1	2
Temp	Half-life	Half-life	Remark
°C	days	days
10	19	9	determined
30	70	44	determined
25	57	35	interpolated

pH 9
	Constituent
	1	2
Temp	Half-life	Half-life	Remark
°C	days	days
10	80	58	determined
30	45	31	determined
25	54	38	interpolated

Executive summary:

The purpose of this study was to determine if the test substance would hydrolyze at environmentally relevant pH values, complying with the OECD Guideline No. 111. The test substance consisted of several constituents of which the two main constituents, based on percentages, were considered to be representative for the test substance (AkzoNobel, 2014). Most probably due to thermal instability of the test substance at 50°C, delivering in inconsistent results, calculations in Tier 2 of the study were confined to the temperatures of 10 and 30°C. An overview of the calculated half-lives for the 2 main constituents of the test substance at the different pH values and tested temperatures is displayed in the table below. Also the interpolated half-lives for the temperature of 25°C are displayed.

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02-06-2017 to 01-09-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

Tier 3

Deviations:

yes

Remarks:

see Principles of method if other than guideline

Principles of method if other than guideline:

There were four modifications to OECD guideline 111:
· No thymol was added to the buffer solutions as, based on microbiological experience, the addition of
thymol as a disinfectant is not necessary because the buffer solutions were sterilized also.
· Test temperature of 12 °C was used. As it was the goal to determine the hydrolysis products at
environmental relevant temperatures.
· In addition to the standard OECD guideline buffers, a natural surface water and demineralized water
containing humic acid was tested. The test was performed above the water solubility, this was
necessary to ensure that sufficient hydrolysis product would be generated, to make identification and
quantification possible.
· Test was conducted using techniques not readily available in contract laboratories and was therefore
not conducted to GLP.

GLP compliance:

yes

Radiolabelling:

no

Analytical monitoring:

yes

Buffers:

Buffer solutions were prepared according to the buffer systems as described in Annex 3 of OECD
guideline 111 (OECD, 2004).

Details on test conditions:

De-ionised water
The de-ionised water used in the study contained less than 10 μg/L of copper (not measured under
GLP), with a conductivity of less than 5 μS/cm and less than 2.0 mg/L NPOC-content.

Materials
Test vessels, buffer solution and test media were sterilised. Test solutions were flushed with nitrogen to
minimise oxidation. Test vessels were kept under dark conditions. A temperature-controlled water bath
was used; temperature was measured using a calibrated thermometer, pH was measured using a pH
meter. The pH buffer 7 was made according to the description of Clak and Lubs, described in Annex 3
of OECD 111 (OECD, 2004). The natural surface water was sampled in the Netherlands

Identification of hydrolysis products (Tier 3)
The sterilised buffer solution pH 7, the surface water and demineralized water containing humic acid
were transferred to volumetric flaks and purged with nitrogen for at least 5 minutes.
A stock solution of the test substance was prepared in dichloromethane and spiked to the buffer
solution and the two other media. The solutions were spiked in 50 mL volumetric flasks at a
concentration of approximately 25 mg/L, not exceeding 1 % (v/v) of solvent. Subsequently 10 mL of the
spiked solutions was transferred to multiple sterile glass test vials. The vials were closed tightly and
placed in a thermostatically controlled water bath in the dark at a temperature of 12 ± 0.5°C. At the
moment the test vials were placed in the water bath, the first sample was taken and analyzed using the
analytical methods described in annex 3. Subsequent samples were taken on different time intervals
and analyzed to determine which and the amount of hydrolysis products formed. Samples were
analyzed directly after sampling in order to prevent further hydrolysis and/or thermal decomposition. In
order to ensure that sufficient hydrolysis product would be generated, to make quantification possible,
the water solubility limit was exceeded.

Duration:

90 d

pH:

7

Temp.:

12 °C

Transformation products:

no

Remarks:

no expected or unknown transformation products were seen to increase in concentration during the course of the study

Details on hydrolysis and appearance of transformation product(s):

At the start of the test, next to the test substance, the following products were already percent; MEK
(methyl ethyl ketone), acetic acid and MEK type 3. These products are also the expected hydrolysis
products. The concentrations of these products were followed during the course of this study, however
as the concentrations of these products did not increase significantly, supported by the lack of new
products being formed and the Trigonox 301 concentration not decreasing significantly, it was
concluded that no hydrolysis products were formed during the 90 day test period at 12 °C.

pH:

7

Temp.:

12 °C

DT50:

> 90 d

Remarks on result:

hydrolytically stable based on preliminary test

Validity criteria fulfilled:

yes

Remarks:

Test temperatures were measured to be within the criteria (12.0 +/- 0.5 oC); the pH value of the buffer pH 7 was measured to be within the criteria (7 +/- 0.1)

Conclusions:

Existing hydrolysis data was brought into question due to the lack of biodegradation and the
discrepancies between the test substance and its analogues. Due to this data being critical to the
interpretation of the persistence of the test material an environmentally relevant data point showing
apparent rapid hydrolysis in existing data (Harlan 2014) was selected for a repeat and identification of
hydrolysis products.
The purpose of this study was to determine the hydrolysis products of 3,6,9-triethyl-3,6,9-trimethyl-
1,4,7-triperoxonane at pH 7 , according to Tier 3 of OECD Guideline No. 111, at a single
environmentally relevant temperature. In addition to the standard buffer solution, the hydrolysis
products were also determined in media containing organic material as this has for some peroxide
groups influenced stability.
No significant increase in hydrolysis products were detected during the course of this 90 day study. The
presence of organic material, addition of an iron complex or elevating the temperature had no effect on
the stability of the test substance.

Executive summary:

No significant increase in hydrolysis products were detected during the course of this 90 day study. The

presence of organic material, addition of an iron complex or elevating the temperature had no effect on

the stability of the test substance.

Description of key information

The registered substance is rather stable at environmental relevant temperatures. This result is confirmed by an OECD 111 Tier 3 test with CAS 24748 -23 -0 that indicates that at a pH value of 7, the half-life (t½) for 3,6,9-Triethyl-3,6,9-trimethyl-1,4,7-triperoxonane at a temperature of 12°C was > 90 days.

Additional information

In Tier 2 of the study initially three test temperatures were used: 10, 30 and 50°C. The tests at 50°C however showed very inconsistent results. This was most probably caused by the thermal instability of the test substance. Therefore it was decided to confine Tier 2 of the study to 10 and 30°C. Results for the different pH values and test temperatures are described below.

Constituent 1: 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane

Constituent 2: 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane,

 

pH 4

10°C: The average half-lives were calculated to be 63 days for constituent 1 and 20 days for constituent 2.

30°C: The average half-lives were calculated to be 13 days for constituent 1 and 10 days for constituent 2.

Using the equations of these interpolation curves, the half lives at a temperature of 25°C were interpolated to be 25 days for constituent 1 and 13 days for constituent 2.

 

pH 7

10°C: The average half-lives were calculated to be 19 days for constituent 1 and 9 days for constituent 2.

30°C: The average half-lives were calculated to be 70 days for constituent 1 and 44 days for constituent 2.

Using the equations of these interpolation curves, the half-lives at a temperature of 25°C were interpolated to be 57 days for constituent 1 and 35 days for constituent 2.

 

pH 9

10°C: The average half-lives were calculated to be 80 days for constituent 1 and 58 days for constituent 2.

30°C: The average half-lives were calculated to be 45 days for constituent 1 and 31 days for constituent 2.

Using the equations of these interpolation curves, the half lives at a temperature of 25°C were interpolated to be 54 days for constituent 1 and 38 days for constituent 2.

From this study it is concluded that the substance is rather stable hydrolytically at environmental relevant temperatures.

These results are confirmed with a test with CAS 24748 -23 -0 (OECD 111 Tier 3) that indicates that at a pH value of 7, the half-life (t½) for 3,6,9-Triethyl-3,6,9-trimethyl-1,4,7-triperoxonane at a temperature of 12°C was > 90 days.