Reaction mass of (2R,5R)-2-isopropyl-5-methylcyclohexanone and (2S,5R)-2-isopropyl-5-methylcyclohexanone

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-16 to 2012-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-confiorm study under GLP without deviations

Data source

Reference

Reference Type:

secondary source

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Menthone/ ISO Menthone Racemic

IUPAC Name:

Menthone/ ISO Menthone Racemic

Test material form:

other: liquid

Details on test material:

- Substance type: organic
- Physical state: liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
The concentrations of the test item 620103 Menthone/ ISO Menthone Racemic were determined in one of the duplicate test medium samples from the nominal test concentrations of 28 mg/L from all sampling times.

- Sampling method:
samples were taken from each treatment just before the start of the first and of the last renewal periods (Days 0 and 3). At the end of these renewal periods (Days 1 and 4), additional samples were taken for determination of the stability of the test item during the renewal periods of 24 hours.
All samples were taken from the approximate center of each aquarium without mixing the test medium.

- Sample storage conditions before analysis:
All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (GLP, Harlan study D53195), the test item is sufficiently stable in the test water under the storage conditions. In one of each of the duplicate samples, acetonitrile was added (10 mL sample + 3 mL ACN) immediately after sampling to stabilize the latter during the storage period. Thereafter, all samples were stored at about -20 °C.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: At the start of the test and before each test medium renewal, the test medium of nominal 28 mg/L was freshly prepared by completely dissolving 304 mg of the test item in 10.85 liter of test water using stirring for 30 minutes at room temperature. The test medium was freshly prepared just before introduction of the fish (= start of the exposure and prior to each test medium renewal).
- Controls: test water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the start of the test and before each test medium renewal, the test medium of nominal 28 mg/L. Based on a prior water solubility study, the water solubility of the test item is 0.57 g/L only (results of Harlan study: D53127).

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: The fish were obtained from a breeding culture at Harlan Laboratories.
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): 2.94 ± 0.15 cm (Mean ± SD)
- Weight at study initiation (mean and range, SD): 0.21 ± 0.05 g (Mean ± SD).
- Method of breeding: During holding the fish were fed with a commercial fish diet.
- Feeding during test: no

ACCLIMATION
- Acclimation period: one week
- Acclimation conditions (same as test or not): test fish were acclimated to the test water and temperature.
- Type and amount of food: During acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet.
- Feeding frequency: no data
- Health during acclimation (any mortality observed): During holding and acclimatization, no fish died in the test fish batch and all fish were healthy.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

1.25 mmol/L CaCO3

Test temperature:

22 °C

pH:

7.1 in all treatments

Dissolved oxygen:

The oxygen concentration was at least 6.6 mg/L

Nominal and measured concentrations:

- Nominal Concentration of Test Item 28 mg/L (threshold approach, limit test)
- Measured: 26.4 - 27.6 mg/L (In the analyzed test medium samples from the start of all analyzed test medium renewal periods, analytically determined concentration were between 97 - 98 %. At the end of the test medium renewal periods, the measured test item concentration was in the range of 94 - 96 %. Thus, the test item was stable in the test medium over the test renewal periods of 24 hours. Therefore the results are based on nominal concentrations.)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask (volatile substance)
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: One 10 liter Erlenmeyer flask (glas) was used for each treatment. The Erlenmeyer flasks were nearly completely filled to keep the air-space in the test flasks as small as possible and were tightly sealed.
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): the test media were renewed daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.13 g fish wet weight per liter test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water was used in the study.
CaCl2 × 2H2O: 1.0 mmol/L; 147 mg/L
MgSO4 × 7H2O: 0.25 mmol/L; 61.5 mg/L
NaHCO3: 0.38 mmol/L; 32.5 mg/L
KCl: 0.038 mmol/L; 2.9 mg/L
- Total organic carbon: Not applicable, as reconstituted water was being used.
- Particulate matter: Not applicable, as reconstituted water was being used.
- Metals: Toxic metals not applicable, as reconstituted water was being used. Detailed mineral composition of test water is given.
- Pesticides: Not applicable, as reconstituted water was being used.
- Chlorine: Not applicable, as reconstituted water was being used.
- Alkalinity: 0.4 mmol/L
- Ca/Mg ratio: 4:1
- Conductivity: Not measured, however the detailed mineral composition of the test water is given in the study report.
- Culture medium different from test medium: yes
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour dark photoperiod, with a 30-minute transition period was used
- Light intensity: 140 to 480 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Sublethal effects (apathy, fish mainly at bottom of aquarium) were observed after 72 and 96 hours of exposure

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test (justification see below)
- Test concentrations:
A limit test was performed in accordance with the threshold approach requiring that the fish test be performed with a single concentration close to or slightly higher than the lowest EC50 value obtained in tests with algae and daphnia. The threshold approach (based on the Guidance on Information Requirements and Chemical Safety Assessment, ECHA (2008), and the OECD Series on Testing and Assessment No. 126, Short guidance on the threshold approach for acute toxicity (2010)) was used to demonstrate, that zebra fish is not the most sensitive species for 620103 Menthone/ ISO Menthone Racemic. The solubility limit was determined in a GLP - pre-experiment under Harlan Laboratory Study D53127.
According to results from pre-experiments (according to the results of pre-experiments under GLP, Harlan Studies D53127 and D53195) and in consultation with the Sponsor the test item is volatile. Therefore, a semi-static test procedure was chosen: the test media were renewed daily to keep the concentrations of 620103 Menthone/ ISO Menthone Racemic in the test media as constant as possible during the test period of 96 hours.

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

- Behavioural abnormalities: At the test item concentration of 28 mg/L, sublethal effects (apathy, fish mainly at bottom of aquarium) were observed after 72 and 96 hours of exposure, but no mortality was observed.
- Other biological observations:
- Mortality of control: no mortality observed
- Other adverse effects control: none observed
- Abnormal responses: none observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test medium. It was a clear solution
- Effect concentrations exceeding solubility of substance in test medium: yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item had no acute toxic effects (based on mortality) on zebra fish up to the nominal concentration of 28 mg/L under the present test conditions.

Executive summary:

The purpose of this study was to determine the acute toxicity to fish of test item. The study followed the protocol as set out in OCED TG 203.

The test item was stable in the test medium over the test renewal periods of 24 hours. The biological results were related to the nominal concentrations of the test item. In the control and test medium with the nominal concentration of 28 mg/L all fish survived until the end of the test. No visible abnormalities were observed at the test fish in the control. At the test item concentration of 28 mg/L, sublethal effects (apathy, fish mainly at bottom of aquarium) were observed after 72 and 96 hours of exposure, but no mortality was observed.

Therefore, the 96-hour NOEC to zebra fish was determined to be < 28 mg/L due to observed sublethal effects. The 96-hour LC0 of to zebra fish was determined to be at least 28 mg/L. The 96-hour LC0 might even be higher than this concentration, but concentrations higher than the lowest EC50 value obtained in tests with algae and daphnia have not been tested in this fish test, in agreement with the guidelines and with the threshold approach. The 96-hour LOEC to zebra fish was determined to be 28 mg/L due to observed sublethal effects. The LC50 and LC100 were clearly higher than 28 mg/L. These values could not be quantified due to the absence of mortality at the tested concentration.

In conclusion, the test item had no acute toxic effects (based on mortality) on zebra fish up to the nominal concentration of 28 mg/L under the present test conditions.