O,O-tert-butyl isopropyl monoperoxycarbonate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No chemical analysis was conducted
In order to maximize exposure test substances were refreshed daily from the (WAF vessels) after 48h (for substance tested via a stock solution).
5 embryos were tested per well in order to maximize statistical significance of this screening data
The test wells were covered during the test with a lid to further reduce loss by volatilization
GLP is not claimed for this data
In addition to the standard guideline criteria for mortality severe malformations that would have without doubt resulted in ultimate embryo mortality have been counted as mortalities for the purpose of this study
Well rinsing took place for all tests to minimize test substance loss to the wells
Substance preparation was conducted as a standard stock solution approach if possible or as a slow stir WAF (water accommodated fraction) if the substance was very poorly soluble or if generation of degradation products was required or if the parent material was a mixture.
Limited screening concentrations aligned with GHS cutoffs were tested
In the third control for the third week of testing a control + Acetone was not tested. Instead two control replicates in DSW only were tested.

Deviations:

yes

Remarks:

See versions/remarks

GLP compliance:

no

Remarks:

GLP is not claimed for this data

Specific details on test material used for the study:

Chemical name: OO-tert-butyl-OO-isopropyl monoperoxycarbonate
CAS no: 2372-21-6
Lot/Batch: Not provided
Hydrolytical stability: Stock sufficiently stable in refrigerator

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Replacement method: Stock 100mg/L Semi Static (new stock for refreshment)
Solubility indication: 2.2 g/L

When the solubility and stability of the material was known to be very low WAF (Water accommodated fraction) solutions were made to prepare the test material.

Slow stir WAF preparations have been shown to be capable of producing a stable concentration of dissolved parent material (as well as stabilizing agent or accumulating degradation products) when loaded in excess of the water solubility and slowly stirred in the same manner as a slow stir water solubility test.

Each WAF vessel was equipped with a Teflon tap that allows only the water accommodated fraction to be transferred without the transfer of undissolved material.

The test material for the WAF prepared substances was weighed accurately and added to each WAF vessel separately. The vessels were then carefully filled with 1 liter of test medium and stirred slowly under sealed conditions at room temperature for approximately 72 hours. Prior to the test the stirring was stopped and all WAF vessels were left to rest for 1 hour after which all of the test wells were rinsed with the appropriate test solution to minimize the absorbance of the test material. The test solutions were then discarded and refilled prior to testing. WAF vessels were then restarted and used for the daily refreshment of the solutions in the same manner.

For test substances that were according to the information provided sufficiently soluble and stable enough to make a stock solution stocks were made in the normal manner and diluted with test medium to reach the desired test concentrations.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

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 2-4 hours old when added to the test solutions. This was confirmed by microscopic observation. Tests were not rescored a few hours after start in order to replace / correct unfertilized eggs due to the number of tests conducted simultaneously.

Embryos arrived 3-4 hpf (hours post fertilization). As soon as the test solutions were made embryos were divided into bulk batches using glass pipettes at the appropriate concentration for each test chemical to prevent delay in exposure caused by preparation time. The wells were then filled with each of the stocks made for each chemical at each concentration. After a maximum of one hour the wells were emptied and refilled, after which the embryos (5 per well) were added. Plates were then incubated for the test duration and observed daily.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

200 mg of CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3 per liter

Test temperature:

26 ºC +/- 1 ºC

pH:

8.2

Dissolved oxygen:

>80% of saturation

Conductivity:

550-650 µS/cm

Nominal and measured concentrations:

Test concentrations: 0.1,1,10,100 mg/L

Details on test conditions:

Tests were conducted with methodology as close as possible to the existing fish test data. If test substances were sufficiently soluble to make a stock of 100 mg/L a standard stock solution and subsequent dilution approach was used for generating the test concentrations. If test substances were poorly soluble, instable and/or mixtures then a 72 hour slow stir technique was used to test the parent substance and/or resulting degradation products at their maximum achievable solubility limits. This has been demonstrated as an effective method for organic peroxides in numerous GLP studies.

Test vessels
Greiner “Bio-One” 24 well sterile suspension culture plates were used as test vessels. Each well contained a maximum volume 3 mL and each plate was closable to reduce evaporation of the test media. Each test plate contained 5 embryos per well totaling 25 embryos per concentration and 20 embryos as an internal plate control. For the controls 30 embryos per control were used.

In general semi-static replenishment was used as with a standard fish test after 48 hours for substances of sufficient stability. Daily replenishment directly from the WAF vessels was carried out for the test materials that were prepared as water accommodated fractions. Testing was otherwise conducted according to the OECD 236 guideline with daily observations.

Every 24 hours, observations were recorded. All atypical effects on the embryo in comparison to the controls were noted. At the end of the exposure period, acute toxicity was determined based on a positive outcome in any of the four lethality observations as detailed in the OECD 236 guideline. The LC50 was then estimated where possible. For other observations there is as yet no finalized guidance on how to interpret any non-lethal findings from this assay. Other non-lethal findings were therefore recorded only at this stage.

Previous work using the OECD 236 guideline for predicting the effects of organic peroxides on adult fish showed a good level of concordance with existing adult fish data. It was noted however during this work that, in order to provide a sufficiently conservative estimation of an adult fish LC50
non-lethal effects should ideally also be included in the prediction. For this reason fish considered to have severe malformations that would ultimately result in their death were considered dead in order to make a worst case LC50 prediction for adult fish.

Acute toxicity is usually expressed as EC20,50,80 (Effect Concentration) values. The ECn values are the concentrations of the test substance showing n% reduction in survival relative to the controls. Depending on the test results obtained, the LOEC (Lowest Observed Effect Concentration) and NOEC (No Observed Effect Concentration) can also be determined. The LOEC is defined as the lowest tested concentration which survival is significantly reduced compared to the control. The NOEC is defined as the highest tested concentration at which survival shows no significant difference relative to the control. Endpoints are usually calculated using a validated statistical software program using the William’s and Trimmed Spearman-Karber / probit methods as appropriate. Dependent on the data generated statistical calculations cannot usually be carried out in screening studies due to the limited concentration range, in which case an estimation of the endpoint range has been made. In the event that the test substance is very poorly soluble, instable or a mixture, loading concentrations; Effect loading concentration (ELn) No observed effect loading rate (NOELR) were used to express the toxicity endpoints.



Test vessels
Greiner “Bio-One” 24 well sterile suspension culture plates were used as test vessels. Each well contained a maximum volume 3 mL and each plate was closable to reduce evaporation of the test media. Each test plate contained 5 embryos per well totaling 25 embryos per concentration and 20 embryos as an internal plate control. For the controls 30 embryos per control were used.

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 2-4 hours old when added to the test solutions. This was confirmed by microscopic observation. Tests were not rescored a few hours after start in order to replace / correct unfertilized eggs due to the number of tests conducted simultaneously.

Test room
The test took place in a temperature controlled incubator set at 26 ºC. The test plates were scored outside of the incubator but were returned as soon as possible after scoring.

Test medium
The test medium Dutch Standard Water (DSW) was used for testing. DSW has a pH of 8.2, conductivity of 550-650 µS/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.

Solution replenishment
Solution refreshment was carried out daily for WAF preparations by removing as much liquid as possible from each well (while avoiding drying of the embryos) using a pipette. After which the appropriate WAF solution was tapped off directly into the test wells. The WAF solutions (in which excess of test material remained visible) were used at the start of the test as well as for all solution replacements.

For test chemicals with sufficient solubility and stability to make stock solutions, old liquid was removed from the wells in the same manner as for WAF preparations. The stocks made at the start of the test were re-used to make new dilutions for the solution replenishment in the same manner as at the start of the test. Stocks were sealed and refrigerated while not in use. Refreshments took place half way through the test after approximately 48 hours. For OO-tert-butyl-OO-isopropyl monoperoxycarbonate (CAS 2372-21-6) a fresh stock was made for solution refreshment.

Addition of test organisms
Embryos arrived 3-4 hpf (hours post fertilization). As soon as the test solutions were made embryos were divided into bulk batches using glass pipettes at the appropriate concentration for each test chemical to prevent delay in exposure caused by preparation time. The wells were then filled with each of the stocks made for each chemical at each concentration. After a maximum of one hour the wells were emptied and refilled, after which the embryos (5 per well) were added. Plates were then incubated for the test duration and observed daily.

 

Reference substance (positive control):

yes

Remarks:

3,4-dichloro aniline

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 10 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The substance was tested for acute toxicity to zebra fish embryos. From this data a prediction for adult fish toxicity was made.
The following validity criteria for all conducted tests were met:

• The overall fertilization rate of all of the eggs was >70%
• Temperature was maintained at 26 ºC using an alarmed incubator
• Survival in 3 of the 3 controls exceeded 90% (when corrected for fertilization loss)
• Hatching in 2 of the three controls exceeded 80% (remaining 3rd control was ended slightly before 96h)


The following validity criteria for all conducted tests were not met:
• Oxygen saturation was not measured in all controls. This had previously been demonstrated to remain acceptable when the test medium is thoroughly saturated before use. It was therefore not measured.
An occasional malformation (1-2 embryos in 60) was observed in the control replicates. This has also been the case in historical controls. This was considered when concluding on test substance related effects.

Results with reference substance (positive control):

Exposure to positive controls resulted in a minimum of 30% mortality in all 3 controls.

Sublethal observations / clinical signs:

Scoring

                       

Abbreviation	Meaning
OK	Okay (not hatched unless (H))
H	Hatched
HOK	Hatched & Okay
IC	Internal Plate Control
DEL	Delayed
BT	Severe tail bend
M	Severe malformation
ED	Edema (usually heart)
C	Coagulated Egg
D	Death/Mortality
Mob	Effects on mobility Reduced/increased
S	Reduced size
CV	Curved tail
DS	Development stalled (Coagulated)
ST	Short tail
T	Tail

 

Test substance (2732-21-6) 96h

 

Test	1	2	3	4	5	6   Not Tested Insufficient Embryos	Predicted Adult Fish LC50 (% mortality)
0.1 mg/L	4HOK 1C	4HOK 1C	4HOK 1C	3HOK 2C	4HOK 1MT*		20
1  mg/L	4HOK 1OK	4HOK 1C	5HOK	3HOK 2C	4HOK 1C		32
10 mg/L	5HOK	5HOK	4HOK 1OK	4HOK 1C	4HOK 1OK		4
100 mg/L	1HD 3C 1D	5HD	5HD	4HD 1C	5HD		100
Plate Control

* Moderate Tail malformation

 

LC₅₀= 10-100 mg/L

 

 

RAW DATA(96h only)

Controls (20/03/17)

Test	1	2	3	4	5	6	Survival %	Malformations Severe %	Hatching pooled %
DSW	3HOK 2OK	2HOK 3OK	4HOK 1C	4OK 1HOK	4HOK 1C	2HOK 3OK	93	0	70
DSW+ ACETONE	2HOK 2OK 1C	4HOK 1C	5HOK	3HOK 2OK	3HOK 1OK 1M	4HOK 1OK	90	3
3,4, DCA	4M 1C	5M	3M 2C	4M 1C	3C 2M	3M 2C	0	70	0

Note:Hatching slightly low due to earlier test termination all unhatched embryos appear normal and are in the process of hatching.

Controls (10/04/17)(96h only)

Test	1	2	3	4	5	6	Survival %	Malformations Severe %	Hatching Pooled %
DSW	5HOK	4HOK 1OK	3HOK 2OK	3HOK 2C	4HOK 1OK	3HOK 1C 1HS	90	0	93
DSW+ ACETONE	5HOK	3HOK 2C	5HOK	5HOK	5HOK	4HOK 1C	90	0
3,4, DCA	4M 1C	3M 2C	4M 1OK	3M 2C	3ED 1OK 1C	2C 3M	7	93	0

 

Controls (15/05/17)(96h only)

Test	1	2	3	4	5	6	Survival %	Malformations Severe %	Hatching Pooled %
DSW I	4HOK 1C	5HOK	4HOK 1OK	3HOK 2C	4HOK 1OK	3OK 1M 1C	83	4	80
DSW II	3HOK 2OK	4OK 1HOK	3HOK 2OK	3HOK 2OK	3HOK 2C	4HOK 1C	90	0
3,4, DCA	2M 3C	2M 3C	2M 3C	2M 3C	3M 2C	3M C	0	46	0

Note: Had DSWI has not been corrected for non-fertilized eggs. Had this been the case survival would also have exceeded 90%.

Validity criteria fulfilled:

yes

Conclusions:

An estimation of the LC50 for adult fish species was made the material tested. Together with existing GLP fish data as well as the existing investigations of FET test applicability for organic peroxides the author considers it possible to reduce animal testing using this data. Either by a weight of evidence approach in conjunction with QSAR calculations or by demonstrating the suitability of read across to an existing GLP fish endpoint of an analogue or otherwise related substance.
The validity criteria were primarily met and where this was not the case this was justified accordingly. The embryo batches were considered of good quality and sufficient for use in the OECD 236 test.
The test substance had a predicted adult fish LC50 of > 10, < 100 mg/L.

Executive summary:

The objective of this study was to screen the effects of the tested chemicals for their effects on newly fertilized zebra fish eggs and hatchlings over an exposure period of 96 hours according to the OECD 236 guideline. Data for each group of organic peroxides has been generated previously and the concordance with fish data was considered acceptable when comparing data to available adult fish test data. Screening tests covering a broader range of test concentrations but covering the familiar GHS cutoffs for aquatic toxicity have therefore been used to allow an indication of the expected toxicity range to fish species. Together with the existing adult fish data for similar substances and by using QSAR calculations it is the intention to fill existing data gaps and support read across with this data as an alternative to additional animal testing.  

The test substance had a predicted adult fish LC50 of > 10, < 100 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, and documentation / justification is limited

Remarks:

ECOSAR typically does not provide reliable quantitative aquatic predictions for organic peroxides (as evidenced by a large experimental REACH database), but it is used to qualitatively compare the trophic levels to estimate their relative sensitivities

Principles of method if other than guideline:

calculation

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 91.137 - < 232.383 mg/L

Remarks on result:

other: ECOSAR v1.11 calculation, Neutral Organic SAR Class (91.137) vs Peroxy Ester Class (232.383)

Sublethal observations / clinical signs:

Calculated short-term toxicity to aquatic invertebrates is 26.744 -53.744 mg/L (Peroxy Esters Class - Neutral Organic SAR Class).

Calculated toxicity to aquatic algae and cyanobacteria is 13.144 - 25.274 mg/L (Peroxy Esters Class - Neutral Organic SAR Class).

Based on these calculations, algae is expected to be the most sensitive species, which is supported by the experimental results.

Validity criteria fulfilled:

not applicable

Conclusions:

The calculated (ECOSAR v1.11) short-term toxicity to fish LC50 (96h) is 91.137 mg/L (Neutral Organic SAR Class) - 232.383 mg/L (Peroxy Ester Class).

Calculated short-term toxicity to aquatic invertebrates is 26.744 -53.744 mg/L (Peroxy Esters Class - Neutral Organic SAR Class).
Calculated toxicity to aquatic algae and cyanobacteria is 13.144 - 25.274 mg/L (Peroxy Esters Class - Neutral Organic SAR Class).

Based on these calculations, algae is the most sensitive species and more importantly, that fish is NOT the most sensitive species, which is supported by the experimental results.

Executive summary:

The calculated (ECOSAR v1.11) short-term toxicity to fish LC50 (96h) is 91.137 mg/L (Neutral Organic SAR Class) - 232.383 mg/L (Peroxy Ester Class).

Description of key information

An estimation of the LC50 for adult fish species was made. The effects on newly fertilized zebra fish eggs and hatchlings over an exposure period of 96 hours according to the OECD 236 were determined. The test substance has a predicted adult fish LC50 of > 10 and < 100 mg/L. 10 mg/L shall be used as worst case for chemical safety assessment. ECOSAR is used as qualitative weight of evidence that fish is not the most sensitive endpoint, algae is the most sensitive endpoint (experimentally and qualitatively from ECOSAR).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

10 mg/L