tert-pentyl 2-ethylperoxyhexanoate

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:

1989-09-19 to 1989-12-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no analytics performed

Justification for type of information:

see cross-referenced read across: supporting information in Section 13.2 attachment

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

1984

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
NA

Analytical monitoring:

not specified

Details on sampling:

NA

Vehicle:

yes

Details on test solutions:

As the test substance had a water solubility of 210 - 240 µg/mL, acetone was used as an organise solvent for the preparation of the stock solution in the preliminary test 2 -10 mg/L) and the definitive test. The stock solution in the definitive test contained 210 mg/L of test substance per litre of acetone.
The selected test concentrations used in the definitive test (1.0 - 2.1 - 4.6 - 10.0 - 21.0 mg/L) were prepared by the addition of the respective amounts of stock solution to the diluting water. By using the acetonic stock solution an acetone concentration of 100 µL/L was obtained for each test concentration and control.

Test organisms (species):

Poecilia reticulata

Details on test organisms:

TEST ORGANISM
- Common name: Poecilia reticulata (guppy)
- Source: Rasbora/Aqualent
- Length at study initiation: ca. 2 cm
- Feeding during test: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

NA

Hardness:

231.4 mg/L CaCO3

Test temperature:

between 24°C and 25°C

pH:

8.2

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

1.0, 2.1, 4.6, 10.0, 21.0 mg test substance/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
As test vessels were used 2 L covered glass aquaria which contained 1 L of test medium. The loading of the test vessels was approximately 0.6 g biomass/L. The surviving fish were counted daily and dead fish removed immediately upon observation.

TEST MEDIUM / WATER PARAMETERS
The diluting water was a synthetic water, Dutch Standard Water, according to NPR 6507 (1980). The deionized water contained not more than 0.01 mg copper per litre and had a conductivity of less than 5 µS/cm. The TOC-content was not more than 2.0 mg/L.


OTHER TEST CONDITIONAS
- Photoperiod: 12 hours of ambient light per day

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

8.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 6.4 and 11.74 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

After 96 hours no mortality was observed at concentrations up to and including 2.1 mg/L (NOEC, no observed effect concentration), whereas 21.0 mg/L produced 100% mortality within 72 hours.
No changes in the behaviour or the appearance of the fish were observed.

The quality criteria of this test have been fulfilled: the mortality in the control was ≤ 10%, the oxygen concentration was > 60 % of the air saturation value throughout the test, except at the concentration of 9.7 mg/L where it was slightly lower.

Results with reference substance (positive control):

No positive control

Reported statistics and error estimates:

see below at " Any other information on results incl. tables"

Sublethal observations / clinical signs:

Mortality in definitive test:

mg test substance/ L	number of surviving animals
	0 h	24 h	48 h	72 h	96 h
control I	10	10	10	10	10
control II (acetone)	10	10	10	10	9
1.0	10	10	10	10	10
2.1	10	10	10	10	10
4.6	10	10	10	9	9
10.0	10	9	8	6	6
21.0	10	1	1	0
temperature °C	25	24	25	25	nd

nd: not determined

Statistical evaluation:

time (d)	LC50 (mg/L)	95% confidence limits
1	14.69	10.65 – 20.27
2	13.28	9.73 – 18.14
3	9.32	6.90 – 12.59
4	8.66	6.39 – 11.74

Validity criteria fulfilled:

yes

Conclusions:

The LC50 (96 h) as calculated was 8.66 mg/L with 95% confidence limits of 6.4 and 11.74 mg/L.

Executive summary:

The closely related substance tert.-Butylperoxy-2-ethylhexanoat was tested in an acute toxicity test with Poecilia reticulata (guppy) under semi-static conditions in accordance with EEC method C.1 (1984) and OECD Test Guideline 203 (1984).

Due to the poor solubility of the test substance in water the test concentrations were prepared using acetone as an organic solvent. Each test concentration contained 100 µL of acetone, as did one control.

After 96 hours no mortality was observed at concentrations up to and including 2.1 mg/L (NOEC, no observed effect concentration), whereas 21.0 mg/L produced 100 % mortality within 72 hours.

The LC 50 (96 h) as calculated was 8.66 mg/L with 95 % confidence limits of 6.4 mg/L and 11.74 mg/L.

No changes in the behaviour or the appearance of the fish were observed.

Reference 2

Endpoint:

fish embryo acute toxicity (FET)

Remarks:

Study conducted adequately to OECD236 guideline but without GLP and chemical analysis in support of read across to existing GLP fish endpoint of a closely related substance.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Considered reliable as supporting evidence but not as stand alone study

Justification for type of information:

Use of the FET endpoint is not currently accepted as a direct replacement for the adult fish test. Hence OECD 236 data is used here in support of read across to existing GLP fish data for a closely related material. In order to justify this FET studies have been conducted and compared with existing fish data for all peroxide groups and have shown to be in the same order of magnitude in all cases for which a reliable test could be conducted. OECD 236 or FET data is therefore considered of sufficient accuracy to allow an estimation of expected adult fish toxicity within the standard GHS cutoffs of 0.1-1, 1-10,10-100 etc. Data is therefore considered valid as supporting evidence.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Current Version

Deviations:

yes

Remarks:

Conducted as a broad screening for proof of principle. No Analysis and non GLP.

Principles of method if other than guideline:

• No chemical analysis was conducted. Test substance was known to be hydrolytically stable. This was considered sufficient for the purpose of effect comparison with existing fish data / screening purposes.
• In order to maximize exposure test substance were refreshed after 48h
• 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 possible volatilization
• GLP is not claimed for this data but test guideline was followed.
• 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 to produce a more conservative LC50.
• Well rinsing took place for all tests prior to addition of embryos to minimize test substance loss to the wells
• Substance preparation was conducted as a standard stock solution approach substance has sufficient solubility for a 20mg/L stock solution.
• Limited screening concentrations aligned with GHS cutoffs were tested to allow LC50 range estimation only

GLP compliance:

no

Remarks:

Test guideline was followed

Specific details on test material used for the study:

Test Chemical

Chemical name : tert-amyl peroxy-2-ethylhexanoate
Peroxide group: Peroxyesters
Cas: 686-31-7
Sample ID : Reach sample 0231 (Lot/Batch: 1204442077) CoA available.
Stablity: Hydrolytically stable
Solubility limit : 17.6 mg/L


Reference substance

The reference substance as indicated below was used for the positive control is required by the test guideline:

Chemical name 3,4-dichloro aniline
Supplier Aldrich
CAS-number 95-76-1
Batch/Lot number 13509KQV
Purity 98%
Solubility Poor
Appearance White crystals

Analytical monitoring:

no

Remarks:

Test was for screening purposes, substance was shown to be hydrolytically stable, wells were covered, wells were pre-rinsed and test substance repleacement took place.

Details on sampling:

N/A

Vehicle:

no

Remarks:

Tested up to solubility limit no vehicle required

Details on test solutions:

The test material was sufficiently soluble to make a stock solution. A stock solution of 20 mg/L was made in test medium by addition and stirring until a homogenious stock resulted. This stock was then used to generate the test concentrations in the standard manner by further dilution in test medium. The stock was sealed and stored in the refrigerator after use and was resused to remake the test solutions after 48 hours when the solutions were replenished. test wells were rinsed with the appropriate solutions allowwed to stand and then were discarded before refilling the wells and adding the test organisms.

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

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

N/A

Hardness:

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 addition of the stocks and continual aeration before being used in the test.

Test temperature:

26ºC

pH:

8.2

Dissolved oxygen:

Saturated before use

Salinity:

No Data

Conductivity:

550-650 µS/cm

Nominal and measured concentrations:

0.1,1,10,20

Details on test conditions:

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.

Reference substance (positive control):

yes

Remarks:

3,4-dichloro aniline as specified in test guideline

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 - < 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks:

1) Coagulation eggs 2) Lack of somite formation 3) Lack of detachment of the tail-bud from the yolk sac 4) Lack of a heartbeat 5) Severe malformations that would result in ultimate embryo death (Additional Criterion to ensure worst case LC50 estimation)

Remarks on result:

other: results are intended to estimate the expected toxicity range for adult fish based on previous work on FET testing and Organic peroxides

Details on results:

Based on the test data from this study and the findings of the applicability studies of the FET assay for organic peroxides (added in justification) the expected adult fish toxicity from the test material is between 1 and 10 mg/L

Results with reference substance (positive control):

Positive control results were guideline compliant at 4 mg/L >30% mortality was observed.

Reported statistics and error estimates:

No statistics applied due to broad spaced concentrations being used for range finding purposes. The intention is an LC50 estimation not exact LC50.

Sublethal observations / clinical signs:

Raw Data

Control data 1

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

Control data 2 

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

Control data 3

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

Test Material

 

Test	1	2	3	4	5	6 % Survival	Predicted Adult Fish LC50 (% mortality)
0.1 mg/L	4HOK 1C	4HOK 1C	2HOK 3C	4HOK 1C	3HOK 1BT 1C	2HOK 3C	32
1  mg/L	4HOK 1OK	4HOK 1OK	4HOK 1C	3HOK 2C	4HOK 1C	3HOK 2C	16
10 mg/L	2C 3M*	4BT 1C	5M*	2M* 1OK 2C	1HOK 4C	5OK	92
20 mg/L	1BT 3HOK 1C	1M* 2HOK 2C	4OK 1HOK	3OK 1M ED* 1HOK	2BT 2C 1M	4OK 1C	44
Plate Control						70

*  Whole fish malformed and in some cases tail missing                    

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

                     

 LC₅₀> 1 -<10 mg/L

 

Validity criteria fulfilled:

yes

Remarks:

Control data regarding hatching,fertilization,survival met the required criteria. Oxygen concentration was however not measured at the end of the test. Due to the excellent control survival this is not considered detrimental

Conclusions:

In order to make a meaningful conclusion from this data the historical data should also be considered to provide sufficient context. In a study with 7 representative organic peroxides 6 of these provided data comparable to available adult fish test data. The remaining substance data was considered as invalid in this case due to the lack of a homogenius stock solution. The conclusion from this study (attached above) was that the concordance with adult fish data appeared good and if sub lethal effects were also considered. The overlap with adult fish data could be improved further still. Hence use of the fish embryo test for estimation of the toxicity range for fish species is considered acceptable as supporting evidence.

During this study a standard OECD236 guideline was applied adapted for screening purposes. Severe sub lethal effects were considered as a mortality to provide a more conservative LC50 estimation. This study indicates that a fish LC50 would be expected in the range 1-10 mg/L. When considering the existing fish test data of analogue substance 3006-82-4 the LC 50 was measured as 8.66 mg/L.

Executive summary:

Non GLP FET study conducted to study guideline OECD236 adapted for screening purposes. Test substance identification provided. Test substance refreshment carried out. Lethal and sub lethal effects recorded. Considered reliable with restrictions when used as supporting evidence/read across support. Not considered suitable for use as a stand alone test.

Description of key information

The closely related substance tert.-Butylperoxy-2-ethylhexanoat was tested for acute toxicity to fish according to EU-method C.1 (1984) and OECD guideline 203 (1984). In this 96-hour semi-static acute toxicity test with Poecilia reticulata (guppy) the LC50 value was 8.66 mg/L. After 96 hours no mortality was observed at concentrations up to and including 2.1 mg/L (NOEC, no observed effect concentration), whereas 21.0 mg/L produced 100 % mortality within 72 hours.  This is supported by an FET study on 686-31-7 indicating that a fish LC50 would be expected in the range 1-10 mg/L. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

8.66 mg/L