cis-2-tert-butylcyclohexyl acetate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a guideline study

Justification for type of information:

The chronic fish toxicity of Verdox is based on read-across from Cyclaprop. The full documentation is presented in the Endpoint summary of Long-term toxicity to fish. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

0.91 mg/L

Basis for effect:

larval development

Remarks on result:

other: read-across from Cyclaprop

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 Feb 2013 to 27 Mar 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Verdox.

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

July 2013

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

1996

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E 1241-05 (Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes)

Version / remarks:

2005

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from one test chamber of each treatment and control group 2, 4, 9, 10, 14 and 16 days prior to test initiation to confirm the operation of the diluter. Water samples were collected from alternating replicate test chambers of each treatment and control group on Days 0, 6, 13, 20, 27 and 33 (test termination) to determine concentrations of the test substance in the test chambers. Additional samples were collected on Day 33 and stored for possible analysis if needed. All samples were collected at mid-depth in the test chambers, placed in glass vials and processed immediately for analysis.

Vehicle:

no

Remarks:

0.1 mL HPLC-grade dimethylformamide/L

Details on test solutions:

Stock solutions were prepared four times during the test. At each preparation, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 25 or 50 mg/mL. Proportional dilutions of the primary stock were made in DMF to prepare additional stock solutions at nominal concentrations. The stock solutions were mixed by inversion and appeared clear and colorless. Stock solutions were stored under refrigerated conditions and fresh aliquots were placed in the syringe pumps every two to four days during the test. The stock solutions were delivered to the diluter mixing chambers (at a rate of 10.0 µL/minute) where they were mixed with dilution water (at a rate of 100 mL/minute) to achieve the desired test concentrations. The solvent control was prepared by injecting HPLC-grade DMF into the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all test item treatment groups was 0.10 mL/L.

Test organisms (species):

Pimephales promelas

Details on test organisms:

- Common name: Fathead minnow
- Source: Chesapeake Cultures, Inc., Hayes, Virginia
- Age: Embryos collected for use in the test were from 10 individual spawns and were <24 hours old when the test was initiated.
- Post-hatch feeding: Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends. Brine shrimp nauplii were obtained by hatching cysts purchased from Brine Shrimp Direct, Ogden, Utah. Fish were not fed for approximately 48 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Hardness:

136 - 148 mg/L as CaCO3

Test temperature:

24.8 - 25.9 °C

pH:

8.0 - 8.3

Dissolved oxygen:

5.0 - 8.2 mg/L (A dissolved oxygen concentration of 4.9 mg/L represents 60% saturation at 25ºC in freshwater)

Conductivity:

353 - 401 µS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 0 (solvent control), 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L.
- Time-weighted mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Emybro cups: Embryos were held in incubation cups constructed from glass cylinders approximately 50 mm in diameter with 425 μm nylon screen mesh attached to the bottom with silicone sealant. The cups were suspended in the water column of each test chamber and attached to a rocker arm. The reciprocating motion of the rocker arm (2 rpm) facilitated circulation of test water around the embryos during incubation.
- Test chambers: The test was conducted in a temperature-controlled environmental chamber designed to maintain the target test temperature throughout the test period. The test chambers were 9-L glass aquaria filled with approximately 7 L of test solution. The depth of the test water in a representative test chamber was 16 cm.
- Type of flow-through: A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (HPLC-grade dimethylformamide) control, and a negative (dilution water) control. Syringe pumps (Harvard Apparatus, Massachusetts) were used to deliver the five test substance stock solutions and HPLC-grade dimethylformamide (DMF) for the solvent control into mixing chambers assigned to each treatment and the solvent control. The syringe pumps were calibrated prior to the test. The stock solutions were diluted with well water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to test initiation and verified at approximately weekly intervals during the test. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and at approximately weekly intervals during the test to ensure that flow rates varied by no more than ±10% of the mean for the four replicates. The diluter flow rate was adjusted to provide approximately five volume additions of test water in each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the end of the test. Periodically during the test, all organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth.
- No. of embryos per vessel: 20
- No. of vessels per concentration: 4
- No. of vessels per control: 20
- No. of vessels per vehicle control: 20
- Biomass loading rate: Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.057 g of fish per liter of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) at the end of the test was
0.30 g fish/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterized as moderately-hard water.
- Metals: Please see 'Any other information on materials and methods incl. tables' for an overview of the metals analysed in the test medium.
- Pesticides: Please see 'Any other information on materials and methods incl. tables' for an overview of the pesticides and organics analysed in the test medium.
- Chlorine: 4.2 mg/L
- Alkalinity: 174 - 182 mg/L as CaCO3
- Ca/mg ratio: 2.6
- Culture medium different from test medium: same

TEST MEDIUM MEASUREMENTS
- Temperature: The target test temperature during the test was 25 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, weekly during the test, and at the end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control replicate using a Fulscope ER/C Recorder, which was verified prior to test initiation and weekly during the test using a liquid-in-glass thermometer.
- Dossilved oxygen and pH: Dissolved oxygen and pH were measured in alternating replicates of each treatment and control group at the beginning of the test, weekly during the test, and at the end of the test. Measurements of dissolved oxygen were made using a Thermo Orion Model 850Aplus dissolved oxygen meter and pH was measured using a Thermo Orion Model 525Aplus pH meter. When 100% mortality occurred in the replicate, measurements of temperature, dissolved oxygen and pH were taken in that replicate and then discontinued.
- Hardness, alkalinity, conductivity: Hardness, alkalinity and specific conductance were measured in alternating replicates of the negative control (dilution water) and the highest concentration treatment group at the beginning of the test, weekly during the test and at the end of the test. Hardness and alkalinity were measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using an Acorn Series Model CON6 Conductivity-Temperature meter.

OTHER TEST CONDITIONS
- Illumination: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight.
- Light intensity: Light intensity at test initiation was 210 lux. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006 light meter.
- Photoperiod: An automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.

EFFECT PARAMETERS MEASURED: embryonic mortality, post-hatching mortality, sub-lethal effects
During the first day of exposure, embryos were observed twice for mortality and eggs with fungus. Thereafter, until hatching was complete, observations of embryo mortality and the removal of dead embryos were performed once daily. When hatching reached >90% in the control groups on Day 5 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were kept in the egg cups until they hatched and were released into the test chamber, or until death of the embryo occurred. During the 28-day post-hatch exposure period, the larvae were observed daily to evaluate the numbers of mortalities and the numbers of individuals exhibiting clinical signs of toxicity or abnormal behavior. From these observations, time to hatch, hatching success, and post-hatch growth and survival were evaluated. Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch survival was calculated as the number of larvae surviving to test termination divided by the total number of embryos that hatched successfully. Post-hatch growth of the fathead minnows was evaluated at the conclusion of the 28-day post-hatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet and dry weights were measured to the nearest 0.1 mg using an analytical balance. Fish were placed in an oven at approximately 60°C for approximately 120 hours to obtain dry weight data.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Range finding studies: In order to select the most appropriate test concentrations, a non-GLP range-finding test was conducted under flow-through conditions for 18 days. The range finding test was conducted at nominal concentrations of 0.081, 0.27, 0.90, 3.0 and 10 mg/L along with a negative control and solvent control (0.10 mL HPLC-grade dimethyformamide/L). At the end of the non-GLP
range-finding test, the effects of the test item on time to hatch, hatching success, survival and mean wet weight for each treatment and control group were determined.
- Results used to determine the conditions for the definitive study: The majority of fathead minnow embryos in the control and treatment replicates hatched on Days 4 and 5 of the test, with no apparent treatment-related effect on time to hatch. Mean percent hatching success in the negative control, solvent control, 0.081, 0.27, 0.90, 3.0 and 10 mg/L treatment groups was 100, 100, 98, 100, 100, 98 and 84%, respectively. Mean percent survival in the negative control, solvent control, 0.081, 0.27, 0.90, 3.0 and 10 mg/L treatment groups was 94, 98, 94, 96, 90, 94 and 0%, respectively. Mean group wet weight in the negative control, solvent control, 0.081, 0.27, 0.90 and 3.0 mg/L treatment groups was 17.5, 21.3, 16.7, 17.2, 21.3 and 12.4 mg, respectively. Based on the results of the preliminary range-finding test, the nominal test concentrations of 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L were selected for the definitive test.

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

0.91 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% C. I.: 0.049 - 1.1 mg/L

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

larval development

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

larval development

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: 95% C. I.: 0.37 - 20 mg/L

Duration:

33 d

Dose descriptor:

EC10

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Remarks on result:

other: 95% C. I.: 0.15 - 12 mg/L

Duration:

33 d

Dose descriptor:

other: EC20

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% C. I.: 0.97 - 1.3 mg/L

Details on results:

The most sensitive endpoint was growth (measured as total length, wet and dry weight). A summary of the survival, clinical observations and reproduction of the test is presented in 'Any other information on results incl. tables'.
- Time to hatch: Daily observations of the embryos indicated that there were no apparent differences in time to hatch between the control groups and any of the test item treatment groups. The majority of fathead minnow embryos in the control and treatment replicates hatched on Days 4 and 5 of the test. Hatching reached >90% in the control groups on Day 5 of the test, at which time the larvae were released to their respective test chambers. A few embryos in the solvent control, 0.20, 0.38, 1.5 and 3.4 mg/L treatment group remained in the incubation chambers until they hatched or died on the Day 6 of the test.
- Hatching success: Hatching success in the negative and solvent control groups was 98 and 96%, respectively. There were no statistically significant differences in hatching success between the negative and solvent control groups. Therefore, the control data were pooled for comparisons with the treatment groups. Hatching success in the pooled control, 0.20, 0.38, 0.80, 1.5 and 3.4 mg/L treatment groups was 97, 95, 94, 90, 95 and 90%, respectively. Fisher’s Exact test indicated that the decrease in hatching success in the 0.80 and 3.4 mg/L treatment groups were statistically significant in comparison to the pooled controls. However, since the percent hatching success in the 0.80 and 3.4 mg/L treatment groups did not exhibit a dose-responsive reduction and exceeded the control criterion for the percent hatching success, the statistically significant differences noted at the 0.80 and 3.4 mg/L treatment groups were not considered biologically meaningful or treatment-related.
- Larval survival: Mortality in one replicate of the 0.20 mg/L treatment group occurred prior to the release of the larvae into the test chamber and was not consistent with the timing of larval mortality in other replicates at this concentration or at other test concentration. Therefore early larval mortality in the replicate of the 0.20 mg/L treatment group was not considered to be treatment related, and the data from this replicate were excluded from the statistical analysis of survival at test end. Larval survival in the negative and solvent control groups was 97 and 99%, respectively. There were no statistically significant differences in larval survival between the negative and solvent control groups. Therefore, the control data were pooled for comparisons with the treatment groups. All larvae in the 3.4 mg/L treatment group were dead by Day 12 of the test. Larval survival in the pooled control, 0.20, 0.38, 0.80, 1.5 and 3.4 mg/L treatment groups was 98, 93, 93, 93, 70 and 0%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 1.5 and 3.4 mg/L treatment groups in comparison to the pooled controls.
- Sub-lethal effects: In general, the majority of the fish in the control groups and in the 0.20, 0.38 and 0.80 mg/L treatment groups that survive to test termination appeared normal, with occasional incidental observations of small or weak fish or a fish with a crooked spine. At the 1.5 and 3.4 mg/L test concentrations sublethal signs of toxicity included erratic swimming, loss of equilibrium and fish lying on the bottom of the test chambers. Fish that were weak or smaller in stature were also noted at a higher frequency of occurrence at these concentrations. The larvae in the 3.4 mg/L treatment group were noted lying at the bottom of the test chambers from Day 0 post-hatch until they were all dead on Day 5 post-hatch.
- Growth: Data from one replicate of the 0.20 mg/L treatment group were excluded from the statistical analyses of growth due to incidental larval mortality in this replicate. Although, a statistically significant reduction in percent survival was noted in the 1.5 mg/L treatment group, the percent reduction was slight; therefore, the growth data from this treatment group was included in the statistical analyses of growth endpoints. There were no statistically significant differences in any of the growth parameters between the negative and solvent control groups. Therefore, the control data were pooled for comparisons with the treatment groups. Dunnett’s one-tailed test indicated statistically significant reductions in total length and wet weight among fish in the 0.80 mg/L treatment group and statistically significant reductions in dry weight in the 0.20 and 0.80 mg/L treatment groups in comparison to the pooled controls. Since the reductions in growth at the 0.20 mg/L treatment group did not follow a dose response pattern, the reductions in growth at the 0.20 mg/L treatment group were not considered to be treatment related. In addition, the reductions in mean total length, wet weight and dry weight in the 0.80 mg/L treatment group from the pooled controls (3.2, 8.8 and 6.4%, respectively) were less than 10% and were not considered to be biologically meaningful.
- Test item solubility: The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.

Reported statistics and error estimates:

Please see 'Any other information on materials and methods incl. tables' for a detailed description of the statistical analyses of the study.

Table: Summary of Hatching Success, Larval Survival and Growth of Fathead Minnows Exposed to the test item

Mean Measured Concentration (mg/L)	Percent Hatching Success	Percent Survival to Day 28 Post-Hatch	Growth Parameters at Day 28 Post-Hatch
			Mean Total Length ± SD (mm)	Mean Wet Weight ± SD (mg)	Mean Dry Weight ± SD (mg)
Negative Control	98	97	24.7 ± 0.22	103.1 ± 3.2	20.5 ± 0.90
Solvent Control	96	99	24.8 ± 0.50	106.1 ± 8.4	20.2 ± 1.4
Pooled Control	97	98	24.7 ± 0.37	104.6 ± 6.1	20.3 ± 1.1
0.2	95	93 (3)	24.2 ± 0.40 (3)	97.0 ± 5.3 (3)	18.3 ± 1.0*(3,4)
0.38	94	93	24.6 ± 0.32	103.7 ± 6.2	20.3 ± 0.66
0.8	90*(1)	93	23.9 ± 0.42*(5)	95.4 ± 2.5*(5)	19.0 ± 0.42*(5)
1.5	95	70*	22.7 ± 0.83	89.4 ± 6.2	19.0 ± 1.5
3.4	90*(2)	0*	--	--	--
EC10 (95% CI)(4)	>3.4	0.91 (0.049 – 1.1)	2.7 (0.37 – 20)	1.3 (0.15 - 12)	NA
EC20 (95% CI)(4)	>3.4	1.2 (0.97 – 1.3)	NA	NA	NA

 * There were statistically significant reductions (p ≤ 0.05) in percent hatching success, survival and growth in comparison to the pooled

controls (percent hatching success and survival using Fisher’s Exact test; growth using Dunnett’s one-tailed test).

1) Since the percent hatching success of the 0.80 mg/L treatment group did not follow a dose-response pattern the statistically significant

reductions found in this treatment group in comparison to the pooled controls was not considered to be biologically meaningful.

2) Since percent hatching success at the 3.4 mg/L treatment group was comparable to the percent hatching success of the 0.80 mg/L treatment

group and exceeded the control criterion of 70%, the statistically significant reduction found was not considered to be biologically

meaningful.

3) Data from Replicate D of the 0.20 mg/L treatment group were excluded from the analysis of survival and growth since the mortality in this

replicate occurred prior to the larvae being released into the growth chamber and was not considered to be related to treatment.

4) Since the mean dry weight of the 0.20 mg/L treatment group did not follow a dose-response pattern, the statistically significant reduction

noted in mean dry weight at the 0.20 mg/L treatment group was not considered to be biologically meaningful.

5) Since the percent reductions in mean total length, wet weight and dry weight of the 0.80 mg/L treatment group from the pooled control

were slight (3.2, 8.8 and 6.4%, respectively), the reductions noted were not considered to be biologically meaningful.

-- = No data due to 100% mortality.

NA = not applicable; since the calculated ECx value was extrapolated beyond the data range used in the calculation or the 95% confidence interval contains zero or was overly wide.

DETERMINATION OF THE NOEC

Since the growth data from the study did not meet the criteria for calculation of the ECx values given in the guideline as shown above, i.e. the 95% confidence interval span the entire range of test concentrations and included zero in the ECx estimated for the dry weight. The estimated ECx values for dry weight were well above the concentrations with available dry weight data since there were too little changes and too few concentrations to distinguish between utility of different model. Consequently, the NOEC was reported for the growth endpoints as recommended by the test guideline.

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on materials and methods incl tables'.

Conclusions:

The 33-d NOEC is 0.80 mg/L in freshwater fish (P. promelas)

Executive summary:

The chronic toxicity to fish was determined in a study according to OECD 210 and in compliance with GLP criteria. In this study fathead minnow embryos (P. promelas), (80 per concentration) were exposed to nominal concentrations of 0 (control), 0 (solvent control), 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L for 33 days under flow-through conditions. The exposure period included a 5-day embryo hatching period, and a 28-day post-hatch juvenile growth period. Analytical confirmation of test concentrations showed that the test concentrations did not remain within ±20% of nominal concentrations throughout the test. Therefore, the mean measured concentrations of <LOQ (control), <LOQ (solvent control), 0.20, 0.38, 0.80, 1.5 and 3.4 mg/L were used for the derivation of the effect values. Observations of the effects of the test item on time to hatch, hatching success, growth (measured as total length, wet and dry weight), survival and sub-lethal effects were recorded throughout the study. The water quality parameters measured at the beginning of the test, weekly during the test and at the end of the test in alternating replicates of the negative control (dilution water) and the highest concentration treatment group were determined to be within acceptable limits. The validity criteria of the test guidelines were fulfilled. Hatching success in the pooled control, 0.20, 0.38, 0.80, 1.5 and 3.4 mg/L treatment groups was 97, 95, 94, 90, 95 and 90%, respectively. Larval survival decreased significantly in the two highest test concentrations in comparison with the pooled control (98, 93, 93, 93, 70 and 0%, respectively). In general, only in the two highest test concentrations, sub-lethal effects were observed. Significant biologically relevant effects on growth were observed in the 1.5 mg/L treatment group (could not be observed in the 3.4 mg/L treatment group due to mortality). As growth was the most sensitive biological endpoint measured in this study, the 33-d NOEC was determined to be 0.80 mg/L, based on growth.

Description of key information

EC10 long-term fish toxicity, of Verdox using read across from Cyclaprop is 0.91 mg/l.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.91 mg/L

Additional information

The long-term fish toxicity of Verdox is based on read-across from Cyclaprop. The read-across justification is presented below:

Aquatic toxicity of Verdox (CAS #20298-69-5) based on read-across from data available for Cyclaprop (CAS #68912-13-0)

Introduction and hypothesis for the analogue approach

Verdox (CAS # 20298-69-5) is the acetate ester of 2-t-butylcyclohexyl alcohol. For this substance no data on the long-term toxicity to fish is available. In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the long-term toxicity to fish of Verdox, the analogue approach is selected because for a closely related analogue, Cyclaprop, reliable long-term toxicity to fish data is available.

Hypothesis: Verdox has similar long-term toxicity to fish as Cyclaprop, as these substances are similar in structures and related properties.

Available experimental information: For the source chemical Cyclaprop data is available from a GLP guideline long-term study with fish. The 33-d EC10 value for Cyclaprop is 0.91 mg/L. The data from this study are reliable without restriction (Klimisch 1).

Target chemical and source chemical

Chemical structures of the target and the source chemicals are shown in the data matrix, including physicochemical properties and available ecotoxicological information.

Purity / Impurities

The minor constituent, trans-Verdox does not indicate a different toxic potential. Other impurities are <1%.

Analogue approach justification:

According to Annex XI 1.5 read-across can be used to replace testing when the similarity can be based on a common backbone and a common functional group.

Analogue selection: For Verdox the substance Cyclaprop is selected as an analogue in view of the structural features and log Kow and for which long-term fish toxicity information is available.

Structural similarities and differences: Verdox and Cyclaprop have hydrocarbon backbones to which an ester functionality is attached. The hydrocarbon backbones of Verdox and Cyclaprop result in the same number of carbons. Verdox has a cyclohexyl ring and a tert-butyl group. Cyclaprop has a bridged cyclohexyl ring and has three carbons and in a ring structure. Though Cyclaprop has an unsaturated bond in this latter ring, this is not altering the electrophilicity of the substance being too far away from the ester. This double bond makes the substance somewhat more planar. The functional group in Verdox is an acetic ester, while Cyclaprop has a propyl ester, which will not affect the electrophilicity of the ester functionality.

Bioavailability: Verdox and Cyclaprop have similar bioavailability based on the similarities in chemical structure, molecular weight (MW) and physicochemical properties. Verdox has a MW of 198, while Cyclaprop has 206. Both are liquids, and despite Verdox having one methyl group less the log Kow is slightly higher but < 0.5:4.75 and 4.4, respectively. Verdox is somewhat more volatile from water compared to Cyclaprop, due to a somewhat higher vapour pressure and lower water solubility. For Verdox maintaining stable concentrations in the aquatic solution was more challenging. 

Mode of Action:Verdox and Cyclaprop have the same mode of action based on both having the ester functionality without adjacent groups that increase this electrophilicity.

Other aquatic endpoint results supporting the read across: In the data matrix the effect data of other aquatic data are tabulated. Based on the available ecotoxicological information on the target and source substance, it can be seen that Verdox and Cyclaprop substances show the same ecotoxicological profile for acute toxicity. Acute toxicity for algae and fish are in the 1-10 mg/l range and Daphnia are slightly less toxic exceeding the 10 mg/l. For algae and Daphnia for both Verdox and Cyclaprop long-term Daphnia information is available, resulting in EC10/NOECs of 0.1-10 mg/l. For long-term fish toxicity the EC10/NOEC are also in the 0.1-1 mg/l range when using the Lethal and Critical Body burden approach as ECHA’s R.7b Appendix 7.8-3 (2017).

Body burden: The Lethal Body Burden (LBB) approach for Verdox is calculated first to see which mMol/kg is applicable for the CBB approach. The used equation for long-term fish toxicity is CBB mMol/kg fish = NOEC mMol/l x BCF. The CBB is estimated to between 0.1 and 1 mMol/kg fish. The LBB (LBB mMol/kg fish =LC50 mMol/l x BCF) calculation for Verdox results in 4.41 mMol / kg fish (using LC50 of 5.6 mg/l, BCF of 156 and a molecular weight of 198). This LBB value shows that for the CBB approach the 0.441 mMol/kg fish can be used (instead of 0.1 or 1mMol/kg fish). The long-term fish NOEC thus becomes 0.56 mg/L (0.441 mMol = NOEC mg/l x 156 (molecular weight of 198).

Uncertainty of the predictions both read across and LBB/CBB approach: The read across for Verdox from Cyclaprop has no uncertainties other than those already presented in the sections. There is no need to convert the EC10 fish to Verdox from Cyclaprop because the MW are similar and the measured log Kows are < 0.5, which is within the experimental error of testing.

The LBB and CBB approaches are most applicable to narcotic type of chemicals. Though Verdox is an ester, the neutral organic approach can still be applied, because its ester is not reactive. The ester has no electrophilic groups adjacent to the ester-bond which can increase the reactivity and thus the aquatic toxicity. Also the ester moiety of Verdox will be quickly removed in the (aquatic) and the key degradate, the corresponding alcohol, can be considered a neutral organic. In view of the reasoning above the uncertainty of the CBB prediction is limited.

The long-term fish toxicity of Verdox derived is considered reliable because via two different lines of reasoning very similar long-term fish toxicity values are derived: 0.91 mg/l via read across and 0.56 mg/l using the LBB/CBB approach both in the same 0.1-1 mg/l range.

Data matrix

The relevant information on physicochemical properties and ecotoxicological characteristics are presented in the data matrix below.

Conclusions for long-term fish toxicity

For Verdox aquatic toxicity information is available for all endpoints except long-term toxicity to fish. For this endpoint read-across is performed from the structural analogue Cyclaprop.When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation, which is in the current document. From the analogue justification section it is shown that Verdox is sufficiently similar to Cyclaprop and a EC10 of 0.91 mg/l is used for risk assessment. This value is in line with the value derived from LBB /CBB approach using the BCF of Verdox (0.56 mg/l).

Final conclusion: The EC10 for ong-term fish toxicity of Verdox results in 0.91 mg/l.

 

Data matrix supporting the read across to Verdox from Cyclaprop

Common names	Verdox	Cyclaprop
Source/target	Target	Source
Chemical structures
CAS no.	20298-69-5	68912-13-0
REACH registration	Registered by IFF	Registered by IFF
EINECS	243-718-1	272-805-7
Empirical formula	C12H22O2	C13H18O2
Molecular weight	198.3	206.28
Phys. Chem data	Measured	Measured
Physical state	Liquid	Liquid
Melting point(^oC)	29.8	< -20
Boiling point(^oC)	232	263.5
Vapour pressure (Pa)	9.72 Pa	0.67
Water solubility (mg/l)	10	57
Log Kow	4.75	4.4
Acute aquatic toxicity
Fish 96-h LC50 in mg/L	5.6 (OECD TG 203)	6.7 (OECD TG 203)
Daphnia 48-h EC50 in mg/L	17 (OECT TG 202)	> 14 (OECD TG 202)
Algae 72-h ErC50 in mg/L	4.2 (OECD TG 201)	2.5 (OECD TG 202)
Long-term aquatic toxicity
Fish 33-d EC10 in mg/L	Read-across	0.91 (OECD TG 210)
Daphnia 21-d EC10 in mg/L	0.99 (OECD TG 211)	1 (OECD TG 211)
Algae 72-h NOErC in mg/L	0.57 (OECD TG 201)	1.9 (OECD TG 201)
Microorganisms 28-d NOEC in mg/L	100 (Ready biodegradability test: OECD TG301F)	100 (read across from Cyclacet (acetic ester), which was tested in OECD TG 209)