1,1,3,3-tetramethylbutyl peroxyneodecanoate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1999-07-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study containing three separate tests conducted to appropriate guideline. Tests conducted above the water solubility limit resulting in non relevant physical effects. In light of new solubility data relevant conclusions can be drawn however.

Justification for type of information:

see read across rationale in Section 13.2

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

Two preparation methods used. Two limit tests and one full test were conducted.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

FIRST LIMIT TEST
Seven fish were exposed to a WAF prepared at a nominal concentration of 100 mg/L and a blank control:
Sampling Frequency: At t=0h and t=96h.
Volume: 500 mL from the approximate centre of the test vessel
StorageThe t=0h samples were stored in a freezer prior to analysis. The t=96h samples were analysed on the day of sampling.

SECOND LIMIT TEST
Seven fish were exposed to a WAF prepared at a nominal concentration of 100 mg/L and a acetone control.
Sampling: Frequency at t=0h, t=24 hand t=96 h.
Volume: 500 mL from the approximate centre of the test vessel.
Storage: The t=0h and t=24h samples were stored in a freezer prior to analysis. The t=96h samples were analysed on the day of sampling.


Final Test
During the final LC50 test samples were taken from all concentrations and the solvent-control for analysis.
Sampling: Frequencyt =0h and at t= 48h.
Volume:10 mL from the approximate centre of the test vessels.
StorageNot applicable, samples were analysed on the day of sampling

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species:Carp (Cyprinus carpio, Teleostei, Cyprinidae) (Linnaeus,1758)
Source:Zodiac, proefacc, "De Haar Vissen", L.U. Wageningen The Netherlands.
Mean length:
First limit test: 2.9 ± 0.24 cm
Second limit test: 2.8 ± 0.26 cm
Final LC50 test: 2.3 ± 0.10 cm
Mean weight:
First limit test: 0.84 ± 0.10 g
Second limit test: 0.77 ± 0.21 g
Final LC50 test: 0.72 ± 0.14 g
Characteristics: Fl from a single parent-pair bred in UV-treated water
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 77

Quarantine Acclimation:At least 12 days after delivery.
Medium: ISO-medium, formulated using MiIIi-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

Ca2+: 80 mg/l
Mg2+: 12 mg/l
Na+: 15mg/l
K+: 3 mg/l
Cr: 145 mg/l
SO4: 249 mg/l
HCO3: 47 mg/l
Hardness is 250 mg/L CaCO3
pH, nitrate and nitrite concentration and ammonia concentration: Measured once a week.
Temperature: every day.
Feeding: Daily with Trouvit.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

96 h

Hardness:

250 mg/L as CaCO3

Test temperature:

20.1-20.6

pH:

7.4-7.9

Dissolved oxygen:

6.5-9.1

Salinity:

N/A

Nominal and measured concentrations:

Nominal: 2.2, 4.6, 10, 28 and 46 mg/L.
Measured: 0.73, 1.22, 1.32, 3.69, 6.62 mg/L

Details on test conditions:

Definitive test concditions

Test duration: 96 hours.
Test type:Semi-static with renewal after 48 hours.
Test vessels: 10 litres all-glass.
Test medium: ISO-medium, aerated until the dissolved oxygen concentration had reached saturation and the pH had stabilised. After aeration the
hardness was 250 as CaCO3 per litre and the pH was approximately 8.
Number of fish: 7 fish per concentration and controls
Loading: 0.72 g fish/litre
Illumination: 16 hours photoperiod daily.
Aeration: The test media were not aerated during the test.
Feeding: No feeding from 48 hours prior to the test and during the total test period.
Introduction of fish: Directly after preparation of the test media.
Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.
WAF's prepared at nominal concentrations of 2.2, 4.6, 10, 28 and 46 mg/L.
Test medium without test substance or other additives (0 mg/L).
Test medium without test substance but with the additive (acetone) used in the treatment of the stock solutions, i.e.(100 mg/L).

Reference substance (positive control):

yes

Remarks:

Acceptable in comparison to historical data

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 6.62 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

Nominal loading (46 mg/L)

Basis for effect:

mortality (fish)

Remarks on result:

other: EC50 not reached.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.62 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

Nominal loading (46 mg/L)

Basis for effect:

mortality (fish)

Details on results:

See any other information for Definitive Test Results.

Results with reference substance (positive control):

PCP EC50 (48h)=0.09 mg/L Historical data showed sensitivity of between 0.1-0.46 mg/L for 96 h. The test batch was considered more sensitive than expected from historical data.

Sublethal observations / clinical signs:

Definitive test results

Refreshment

Concentrations WAF	0	2h	24	48	72	96
2.2	7	7	7	7	7	7
4.6	7	7	7	7	7	7
10	7	7	7	7	7	7
28	7	7	7	7	7	7
46	7	7	7	7	7	7
Blank Acetone	7	7	7	7	7	7

No mortality up to 46 mg/L (loading) equal to 6.62 mg/L (measured initial)

Validity criteria fulfilled:

yes

Conclusions:

Testing above solubility is of little environmental relevance and creates misleading data difficult to interpret and use for risk assessment. In this
study the LC50 was not reached at the highest concentration (6.62 mg/L) This is now known to be well in excess of the solubility limit. The NOEC
was also determined at a measured initial concentration of at least (6.62 mg/L) It is therefore safe to assume that this substance would cause no
effects to Fish species at its solubility limit (2.8 mg/L) in water.

Executive summary:

GLP study containing three separate tests , to appropriate guideline. Tests conducted above the water solubility limit resulting in non relevant physical effects. In light of new solubility data relevant conclusions can be drawn however.

Reference 2

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP study performed in accordance to OECD 236

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

See method below

GLP compliance:

no

Remarks:

Study performed in a GLP facility, but not under GLP.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A stock solution in the standard manner was not possible with this material due to the poor solubility of the test substance and likely hood of degradation products being formed. For this reason a WAF approach was used to allow stabilization at the water solubility limit of the parent substance and any other water soluble degradation components that may accumulate during the stirring period. GLP studies had indicated that 6 days stirring was sufficient to measure an increase in the main degradation product and still maintain the parent material at its solubility limit.

The test material was weighed out on a cover glass with an analytical balance. Test material was then loaded to 1 liter of test medium. These solutions were stirred slowly for 6 days.

Stirring speed was sufficient for a slight movement at the water surface but not sufficient to form emulsions or suspensions of the test material.
The cover glass was shattered after addition to the WAF vessel to ensure maximum surface area for the test material to dissolve.

After the 6 day stirring period the agitation was stopped and each WAF was left for one hour to rest. The water accommodated fraction was then tapped directly from the WAF vessels for pH measurements and to fill the well plates as described above.

New WAF solutions were not required for the solution replenishment after 24, 48 and 72 hours. GLP studies had demonstrated stability of the dissolved parent material and accumulation of the main degradation product during this timeframe. See references.

Agitation of the original WAFs was restarted for reuse for in subsequent solution replacements.

Test organisms (species):

other: Danio Rerio embryos

Details on test organisms:

Fertilized zebra fish wild type embryos were sourced at Wageningen UR Animal sciences group 6708 WG Wageningen The Netherlands. Fertilized embryos were between 2-3 hours old when added to the test solutions. This was confirmed by microscopic observation.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

96 h

Test temperature:

The temperature should was maintained at 26ºC +/- 1 ºC during the test.

pH:

The pH of the WAF solutions were measured at the start of the test and the pH was adjusted to that of the control with NaOH or HCL if the pH differed more than 0.5 a pH unit.

Dissolved oxygen:

Oxygen was not measured. All test medium was aerated continually prior to use and was not considered necessary.

Nominal and measured concentrations:

Only nominal WAF concentrations.

Details on test conditions:

Test medium
The test medium Dutch Standard Water (DSW) was used for testing. DSW has a pH of 8.2, conductivity of 550-650µs/cm, and contains: 200 mg of CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3 per liter. The water was made by an automatic dosing system and aerated before being used in the test.

Preperation of WAF solutions
A stock solution in the standard manner was not possible with this material due to the poor solubility of the test substance and likely hood of degradation products being formed. For this reason a WAF approach was used to allow stabilization at the water solubility limit of the parent substance and any other water soluble degradation components that may accumulate during the stirring period. GLP studies had indicated that 6 days stirring was sufficient to measure an increase in the main degradation product and still maintain the parent material at its solubility limit.

The test material was weighed out on a cover glass with an analytical balance. Test material was then loaded to 1 liter of test medium. These solutions were stirred slowly for 6 days.

Stirring speed was sufficient for a slight movement at the water surface but not sufficient to form emulsions or suspensions of the test material.
The cover glass was shattered after addition to the WAF vessel to ensure maximum surface area for the test material to dissolve.

After the 6 day stirring period the agitation was stopped and each WAF was left for one hour to rest. The water accommodated fraction was then tapped directly from the WAF vessels for pH measurements and to fill the well plates as described above.

New WAF solutions were not required for the solution replenishment after 24, 48 and 72 hours. GLP studies had demonstrated stability of the dissolved parent material and accumulation of the main degradation product during this timeframe.

Agitation of the original WAFs was restarted for reuse for in subsequent solution replacements.

Reference substance (positive control):

yes

Remarks:

3,4-dichloro aniline Aldrich

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Results was also monitored for 24, 48 and 72 hours. 96 hours results most relevant and hence reported above.

Results with reference substance (positive control):

The positive control embryos developed abnormalities in 100% of the embryos visible from 24 hours onwards. After 96 hours 100% mortality was determined based on the somite formation criteria. In addition positive controls showed reduced movement, severe edema and multiple malformations.

Sublethal observations / clinical signs:

At 10 mg/L loading (that typically generates a dissolved concentration of up to 36µg/L measured in GLP studies) no differences to the negative controls was observed based on any of the criteria set in the test guideline.

 

Previous GLP studies using the same preparation techniques have demonstrated that due to WAF being loaded far in excess of its water solubility limit that the soluble fraction of the parent material remains present despite the soluble fraction itself being instable. The daily refreshment of the test wells will have ensured the maximum possible exposure to the test substance over the test period.

 

The soluble fraction of the test substance can be concluded as non-toxic to zebra fish embryos at its maximum achievable solubility in test medium in a semi static 96 hour test with daily refreshment. 

Validity criteria fulfilled:

yes

Conclusions:

The following validity criteria for the test were met:

• The fertilization rate of the embryos in the batch tested was > 70%
• The temperature should was maintained at 26ºC +/- 1 ºC during the test
• Exposure to the positive control should did result in a minimum of 30% mortality at the end of the test
• Hatching rate in the negative control should be > 80% at the end of the test


The following validity criterion was not met:

• The oxygen concentration in the control at the end of the test was not measured. The control survival exceeded the validity requirements and hence detrimental shortage of oxygen could have occurred. This is not expected to negatively influence

Executive summary:

A non GLP test was performed in accordance to OECD 236. Due to the low water solubility of the parent, the embryos were exposed to a WAF as desribed in the RSS. A loading of 10 mg/L test material did not result in any significant differencies to Danio rerio embryos. The soluble fraction of th test substances can therefore be concluded as non-toxic to zebra fish embryos at its maximum achievable solubility in the test medium in a semi static 96 hour test with daily refreshment. 

Description of key information

Analogous substance CAS 22288 -41 -1 showed no toxicity up to the maximum water solublity, therefore LL50 > ws. a FET (OECD 236) was performed with CAS 22288 -41 -1 and the registered substance to show similarity and to strengthen the read across. A loading of 10 mg/L test material did not result in any significant alterations to Danio rerio embryos for both substances. This concentration is far above the waters solubility of the substance. The results support that the substance is unlikely to be toxic to fish.

Additional information

A short-term toxicity study according to OECD 203 with 1,1,3,3-tetramethylbutyl peroxypivalate (CAS 22288-41 -1) is available showing no toxicity up to the water solubility limit.

A fish embryo test with the similar substance 1,1,3,3-tetramethylbutyl peroxypivalate (CAS 22288-41 -1) and the registered substance (CAS 51240 -95 -0) was performed as supporting evidence for read across.

A non GLP test was performed according to the fish embryo acute toxicity (FET) test, OECD 236, adopted 26 July 2013. A stock solution in the standard manner was not possible with this material due to the poor solubility of the test substance and likely hood of degradation products being formed. For this reason a WAF approach was used, as described in the RSS, to allow stabilization at the water solubility limit of the parent substance and any other water soluble degradation components that may accumulate during the stirring period. GLP studies had indicated that 6 days stirring was sufficient to measure an increase in the main degradation product and still maintain the parent material at its solubility limit. Test solutions were refreshed daily.

A loading of 10 mg/L test material did not result in any significant effects to Danio rerio embryos. The soluble fraction of the test substances can therefore be concluded as non-toxic to zebra fish embryos at its maximum achievable solubility in the test medium in a semi static 96 hour test with daily refreshment.

Conducting a new fish test would not add any new information for either classification or risk assessment, and hence, the study is waived.