Reaction mass of 7,7-dimethyl-2-methylidenebicyclo[2.2.1]heptane and (1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane and (1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane and (1S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Test method according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens".

Qualifier:

according to guideline

Guideline:

other: US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens"

Deviations:

not specified

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead Minnows
- Source: Animals were hatched and reared at BMRL or obtained from the EPA's Environmental Research Laboratory, Gulf Breeze, FL.
- Age at study initiation (mean and range, SD): 14-28 days old posthatch
- Length at study initiation (length definition, mean, range and SD): 8 - 15 mm standard length
- Weight at study initiation (mean and range, SD):
- Method of breeding:
- Maintenance of the brood fish:

ACCLIMATION
- Acclimation conditions (same as test or not): Flowing, filtered (10-µm) seawater
- Type and amount of food/Feeding frequency: Fish were fed 24-h Artemia salina nauplii (San Francisco Bay Brand) daily until they were used as test animals.
- Health during acclimation (any mortality observed): Fish mortality during the 48-h period immediately before each test was less than 3% and all fish tested appeared to be in good condition.

FEEDING DURING TEST: No

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

25-31 ºC

Salinity:

1.0-3.1 %

Nominal and measured concentrations:

No data on test concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Tests were conducted in either 4-L glass jars that contained 3 L of test solution or 19-L glass jars that contained 15 L
- Aeration: No
- No. of organisms per vessel: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All dilution water was filtered (5-µm), natural seawater of ambient salinity.
- Intervals of water quality measurement: The dissolved oxygen concentration was measured in each test container at initiation of testing and daily thereafter; pH was measured in the control and the low and high test concentrations at the initiation and after 96 h of testing.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality was recorded at 24, 48, 72, and 96 h.

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.6-2.2 ppm)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.6-2.4 ppm)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.6-2.4 ppm)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.0-3.2 ppm)

Reported statistics and error estimates:

Based on the results of the tests, 24-, 48-, 72-, and 96-h LC50's and their 95% confidence limits were calculated when possible. Calculations were performed with a Hewlett Packard Model 9815A calculator programmed (STEPHAN 1977 and 1978 personal communication) to scan the data base and to select the most appropriate of three statistical methods in the following order: moving average angle analysis, probit analysis, or binomial probability. The method selected was determined by the characteristics of the data, that is, the presence or absence of 0% and 100% mortality and the number of concentrations in which >0%<100% mortality occurred. The computer scanned the data, identified the most suited method, and performed the analyses. No correction was made for control mortality.

Sublethal observations / clinical signs:

The LC50 (96 h) was 1.9 ppm (basis for effect: mortality). The NOEC was 1.0 ppm.

Validity criteria fulfilled:

not specified

Remarks:

(No data on control results. There is no evidence that test substance concentration had been maintained throughout the test period).

Conclusions:

The LC50 (96 h) was 1.9 ppm (basis for effect: mortality).

Executive summary:

A marine toxicity static test were conducted at EG&G Bionomics Marine Research Laboratory (BMRL) with sheepshead minnows (Cyprinodon variegatus). Test method was according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". Camphene was tested for 96 hours at 25 -31 ºC. Mortality was recorded at 24, 48, 72, and 96 h. The 96 h-LC50 was 1.9 ppm (basis for effect: mortality). The NOEC was 1.0 ppm.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

other: US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens"

Deviations:

not specified

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

Cyprinodon variegatus

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Nominal and measured concentrations:

No data on test concentrations

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% confidence limits: 1.6-2.2 ppm)

Validity criteria fulfilled:

not specified

Conclusions:

The LC50 (96 h) was 1.9 ppm (basis for effect: mortality).

Executive summary:

A marine toxicity static test was conducted with sheepshead minnows (Cyprinodon variegatus). The 96 h-LC50 was 1.9 ppm (basis for effect: mortality) with 1.6 -2.2 ppm 95% confidence limits.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.9 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Read-across from an analogue

Basis for effect:

mortality (fish)

Remarks on result:

other: read-across from an analogue for which LC50 (96 h) = 1.9 mg/L (95% confidence limits: 1.6-2.2 ppm)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Read-across from an analogue

Basis for effect:

mortality (fish)

Remarks on result:

other: read-across from an analogue for which NOEC (96 h) = 1.0 mg/L

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Read-across from an analogue

Basis for effect:

mortality (fish)

Remarks on result:

other: read-across from an analogue for which LC50 (72 h) = 2.0 mg/L (95% confidence limits: 1.6-2.4 ppm)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Read-across from an analogue

Basis for effect:

mortality (fish)

Remarks on result:

other: read-across from an analogue for which LC50 (48 h) = 2.0 mg/L (95% confidence limits: 1.6-2.4 ppm)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

1.8 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Read-acros from an analogue

Basis for effect:

mortality (fish)

Remarks on result:

other: read-across from an analogue for which LC50 (24 h) = 1.8 mg/L (95% confidence limits: 1.0-3.2 ppm)

Validity criteria fulfilled:

not applicable

Conclusions:

Based on the read-across approach from the analogue camphene, the LC50 (96 h) of reaction mass was determined to be 1.9 ppm (basis for effect: mortality).

Executive summary:

A marine toxicity static test were conducted at EG&G Bionomics Marine Research Laboratory (BMRL) with sheepshead minnows (Cyprinodon variegatus). Test method was according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". Camphene was tested for 96 hours at 25 -31 ºC. Mortality was recorded at 24, 48, 72, and 96 h. The 96 h-LC50 was 1.9 ppm (basis for effect: mortality). The NOEC was 1.0 ppm. Based on these results, the read-across approach was applied and the 96 h-LC50 of the reaction mass was determined to be 1.9 ppm (basis for effect: mortality) and the NOEC 1.0 ppm.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.9 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Read-across from an analogue

Basis for effect:

mortality (fish)

Remarks on result:

other: read-across from an analogue for which LC50 (96 h) = 1.9 mg/L (95% confidence limits: 1.6-2.2 ppm)

Validity criteria fulfilled:

not applicable

Conclusions:

Based on the read-across approach from the analogue camphene, the LC50 (96 h) of reaction mass was determined to be 1.9 ppm (basis for effect: mortality).

Executive summary:

A marine toxicity static test was conducted with sheepshead minnows (Cyprinodon variegatus). The 96 h-LC50 was 1.9 ppm (basis for effect: mortality) with 1.6 -2.2 ppm 95% confidence limits. Based on these results, the read-across approach was applied and the 96 h-LC50 of the reaction mass was determined to be 1.9 ppm (basis for effect: mortality).

Reference 5

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2018 - 19 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0 (vehicle control) and 50 mg/L
- Sampling method: A known volume of sample (10 mL) was taken in a separating funnel and to this 30 mL of dichloromethane was added.
- Sample storage conditions before analysis: not applicable

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of 2500 mg test item was made up to 5 mL of acetone to obtain nominal concentration of 500.0 mg/mL (Stock A). Volume of 400 µL from the stock A was diluted to 4 L with RO water in fish tank to obtain the nominal test concentration of 50.0 mg test item/L.
- Controls: RO water and RO water + acetone
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1mL/L

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Fish-O-Fish-Aquarium
- Length at study initiation: 1.4 ± 0.2 cm

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: "Gemma Micro 500 ZF”, Manufactured by Trouw France SA, 52 ter, Rue Jacob, 02140 Vervins, France.
- Feeding frequency during acclimation: daily
- Health during acclimation (any mortality observed): No signs of physical or behavioural abnormalities or mortalities of fish were observed.

FEEDING DURING TEST
None during test period

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

85.4 ± 5.4 mg CaCO3/L

Test temperature:

initial= 21.7 ± 0.3ºC; final= 21.7 ± 0.2ºC

pH:

initial= 7.34 ± 0.12; final = 7.54 ± 0.22

Dissolved oxygen:

initial=90.4 ± 0.9 %; final=87.8 ± 1.2 %

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0 (vehicle control) and 50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass tank
- Material, size, headspace, fill volume: glass, 8 L capacity, 4 L fill volumen
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): 24 h (semi-static)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): one
- No. of vessels per vehicle control (replicates): one
- Biomass loading rate: maximun 0.6 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO water
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen content of the test media were measured daily before and after each renewal of the media with freshly prepared test solution. Total hardness was analysed daily for each renewal of the diluent water during the study period.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light and 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :Fish were observed at 3, 6, 24, 48, 72 and 96 h of exposure. Recorded parameters included behavioural responses, clinical signs and mortality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Justification for using less concentrations than requested by guideline: no mortality found in a preliminary range finding study.
- Range finding study
- Test concentrations: 0.0 (control), 0.0 (vehicle Control), 0.1, 1.0, 10.0 and 50.0 mg test item/L
- Results used to determine the conditions for the definitive study: The cumulative percent mortalities at 96 h observed were 0, 0, 0, 0, 0 and 0 at the test concentrations of 0.0 (control), 0.0 (vehicle Control), 0.1, 1.0, 10.0 and 50.0 mg test item/L, respectively. Based on the results of this preliminary range finding study, the main study was restricted to limit study. The limit study was conducted with single test concentration of 50.0 mg/L along with control and vehicle control.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Fish exposed to 0.0 (control), 0.0 (vehicle control) and 50.0 mg/L did not exhibit any treatment related behavioral response
- Mortality of control: No mortality was observed over a period of 96 h in the control and vehicle control groups

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

As no mortality was observed at the test concentration of 50.0 mg/L, probit analysis was not carried out.

Sublethal observations / clinical signs:

Table 1: Mortalities

Group	Test Concentration (mg/L)	Percentage Mortality of Fish at						Cumulative Mortality (%) at 96 h
		3 h	6 h	24 h	48 h	72 h	96 h
G1	0.0 (Control)	0	0	0	0	0	0	0
G2	0.0 (Vehicle control)	0	0	0	0	0	0	0
G3	50.0	0	0	0	0	0	0	0

Table 2: Behavioural response

Group	Test Concentration (mg/L)	N^oof Fish Used	Behavioral Response observed at
			3 h	6 h	24 h	48 h	72 h	96 h
G1	0.0 (Control)	10	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
G2	0.0 (Vehicle control)	10	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
G3	50.0	10	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)

Behavioral Response: 1 = Normal

Note: Figures presented outside parentheses refer the clinical symptom and inside parentheses refer the total number of fish

Table 3: Mean of Physico-Chemical Parameters of Test Media

Group	Test Concentration (mg/L)	pH				Temperature (°C)				Dissolved Oxygen (%)
		Initial		Final		Initial		Final		Initial		Final
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
G1	0.0 (Control)	7.26	0.07	7.48	0.15	21.6	0.3	21.5	0.2	91.6	1.2	87.5	2.5
G2	0.0 (Vehicle control)	7.33	0.09	7.55	0.19	21.7	0.2	21.7	0.1	91.5	0.9	88.1	1.4
G3	50.0	7.34	0.12	7.54	0.22	21.7	0.3	21.7	0.2	90.4	0.9	87.8	1.2

Key: Initial = 0 h, Final = 24 h, SD = Standard deviation

Table 4: Total Hardness (mg/L as CaCO3)

Total Hardness (mg/L)	Mean	SD
	85.4	5.4

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10%; semi-static test conditions; dissolved O2 concentration > 60% of air saturation value; concentration of the substance >80% of the nominal concentration)

Conclusions:

In a short-term toxicity test to zebrafish, Danio rerio, the 96h-LC50 of the test substance was found to be greater than 50.0 mg/L.

Executive summary:

An acute aquatic toxicity study with zebrafish, Danio rerio, was conducted on test item according to OECD guideline 203, following GLP. A preliminary range finding study was conducted with test concentrations of 0.0 (control), 0.0 (vehicle Control), 0.1, 1.0, 10.0 and 50.0 mg/L for a period of 96 h with ten fish in each group. Based on no mortalities found in this preliminary study, the main test was restricted to limit study conducted with single test concentration of 50.0 mg/L. Reverse osmosis (RO) water was used as test medium. In the previous solubility study the test item was found to be insoluble in RO water and acetone was selected as the vehicle. Thus, the main test was conducted with test concentrations of 0.0 (control), 0.0 (vehicle Control) and 50.0 mg/L for a period of 96 h in semi-static conditions with ten fish in each group. The test solutions were changed every 24 h with freshly prepared solutions. The test solutions were not aerated during the exposure. A validated analytical method based on gas chromatography was used to monitor the concentration and stability of the main active ingredients in the test solution at 0 and 24 h of first renewal during main study. The active ingredient concentration in the test solution was within acceptable limit (> 80% of nominal concentration). All validity criteria were fulfilled. No mortality and no treatment related behavioural response were observed over a period of 96 h at the test concentration of 50.0 mg/L as well as in the control and vehicle control groups. Thus, LC50 (96 h), NOEC and LOEC to fish of the test substance were found to be greater than 50.0 mg/L.

 

 

 

Description of key information

Key study. Test method according to OECD 203, GLP study. The 96h-LC50 of the test substance to zebrafish, Danio rerio, was found to be greater than 50.0 mg/L.

Supporting studies: Read-across approach. camphene. Test method according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". Based on the read-across approach from the analogue camphene, the LC50 (96 h) of reaction mass to sheepshead minnows (Cyprinodon variegatus) was determined to be 1.9 ppm.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

50 mg/L

Additional information

Key study. An acute aquatic toxicity study with zebrafish, Danio rerio, was conducted on test item according to OECD guideline 203,following GLP. A preliminary range finding study was conducted with test concentrations of 0.0 (control), 0.0 (vehicle Control), 0.1, 1.0, 10.0 and 50.0 mg/L for a period of 96 h with ten fish in each group. Based on no mortalities found in this preliminary study, the main test was restricted to limit study conducted with single test concentration of 50.0 mg/L. Reverse osmosis (RO) water was used as test medium. In the previous solubility study the test item was found to be insoluble in RO water and acetone was selected as the vehicle. Thus, the main test was conducted with test concentrations of 0.0 (control), 0.0 (vehicle Control) and 50.0 mg/L for a period of 96 h in semi-static conditions with ten fish in each group. The test solutions were changed every 24 h with freshly prepared solutions. The test solutions were not aerated during the exposure. A validated analytical method based on gas chromatography was used to monitor the concentration and stability of the main active ingredients in the test solution at 0 and 24 h of first renewal during main study. The active ingredient concentration in the test solution was within acceptable limit (> 80% of nominal concentration). All validity criteria were fulfilled. No mortality and no treatment related behavioural response were observed over a period of 96 h at the test concentration of 50.0 mg/L as well as in the control and vehicle control groups. Thus, LC50 (96 h), NOEC and LOEC to fish of the test substance were found to be greater than 50.0 mg/L.

Supporting studies: Read-across approach. camphene. A marine toxicity static test were conducted at EG&G Bionomics Marine Research Laboratory (BMRL) with sheepshead minnows (Cyprinodon variegatus). Test method was according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". Camphene was tested for 96 hours at 25 -31 ºC. Mortality was recorded at 24, 48, 72, and 96 h. The 96 h-LC50 was 1.9 ppm (basis for effect: mortality). The NOEC was 1.0 ppm. Based on these results, the read-across approach was applied and the 96 h-LC50 of the reaction mass was determined to be 1.9 ppm (basis for effect: mortality) and the NOEC 1.0 ppm.