Reaction Mass of Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.R,1R,6S)- and Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.S,1R,6S)-

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:

key study

Study period:

from 2017-09-02 to 2017-09-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on Jun 07th - Jun 10th, 2016 & Jul 13th. 2016 / signed on 03.01.2017

Specific details on test material used for the study:

- Water solubility: 2,00 mg/L (20°C, pH 5,8)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and limit concentration of 1.50 mg/L (nominal)
- Sampling method: The limit concentration and the control were analytically verified in all fresh and aged test media.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler), if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 1.50 mg/L test item was prepared by adding 1.67 µL/L test item (relative density considered) to dilution water and stirring slowly for 48 h at room temperature. The saturated solution was taken from the homogenous phase and was used for testing.
- Controls: Dilution water without test item subjected to the same test conditions.
- Evidence of undissolved material: No undissolved test item was observed during the test.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Length at study initiation: 2.69 cm (average)
- Weight at study initiation: 0.2 g (average)
- Holding: Fish were held at the test facility at 23 ± 2 °C and diffuse light (7 - 750 lux, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80% of the air saturation value.

ACCLIMATION
- Acclimation period: At least 12 d
- Acclimation conditions: Same as test
- Feeding during acclimation: Fish were fed Sera Vipan (Sera GmbH, Heinsberg Germany) in 4% of the fish body weight, 3 times per week
- Health during acclimation: No mortality was observed, no disease treatment was necessary.

FEEDING DURING TEST
Fish were not fed during the test. Feeding was stopped 24 h before test start.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

58 mg/L CaCO3

Test temperature:

23.0 - 23.9 °C (control)
23.6 - 24.9 °C (treatment)

pH:

7.14 - 7.75 (control)
7.34 - 7.66 (treatment)

Dissolved oxygen:

74 - 98% oxygen saturation (control)
83 - 99% oxygen saturation (treatment)

Conductivity:

138 µS/cm

Nominal and measured concentrations:

Control, and 1.50 mg/L (nominal)
< LOQ , and 0.999 mg/L (geom. mean measured)
See Table 6.1.1/3 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 13 L glass aquaria (25.5 * 18 * 28 cm), filled with 8 L of dilution water (depth of water: 17 cm) and covered with a floating lid (e.g. aluminium foil).
- Aeration: None
- Renewal rate of test solution: 24 h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 7
- No. of vessels per control (replicates): 7
- Biomass loading rate: 0.18 g fish/L test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin, filtered on activated charcoal and aerated for min. 24 h to remove chlorine.
- pH: 6.0 - 8.5
- Alkalinity: 0.6 mmol/L
- Acidity: 0.1 mmol/L
- Conductivity: 138 mS/cm
- Culture medium different from test medium: Dilution water same as holding water.
- Intervals of water quality measurement: The pH value, temperature and oxygen saturation were measured in all test vessels at the start of the test and at the end of the test and every 24 h and after renewal of the test media from fresh and old test media. Total hardness of the water was determined at the beginning in the control. Water temperature was recorded once per hour. Light intensity on the surface of the test aquaria was measured at the start of exposure.

OTHER TEST CONDITIONS
- Photoperiod: 16 h photoperiod daily
- Light intensity: 56 lux (mean value)

EFFECT PARAMETERS MEASURED:
- Mortality: After 3, 24, 48, 72, and 96 h
- Size and weight: After 96 h (control fish)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Threshold approach with one limit concentration of 1.50 mg/L (nominal, saturated solution)
- Range finding study : No range-finding study was conducted but a stability test was carried out with the saturated solution with a nominal loading rate of 1.50 mg/L to determine the optimal stirring time for maximum concentration.
- Results used to determine the conditions for the definitive study: No range-finding study was conducted. The threshold concentration is based on the toxicity data of an alga and a daphnid toxicity study.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.999 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Details on results:

See Tables 6.1.1/1 and 6.1.1/2 in "Any other information on results incl. tables".
- Behavioural abnormalities: All fish were affected by sub-lethal effects. Quiescence was observed after 3 h. Missing escape reflex was recorded after 24 and 48 h. Slowed down and unregular breathing was recorded at 48, 72, and 96 h. Lethargy was recorded after 72 and 96 h.
- Mortality of control: 0%
- Other adverse effects control: None, normal behaviour throughout the test.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No undissolved test item was observed.
- Effect concentrations exceeding solubility of substance in test medium: No

Sublethal observations / clinical signs:

Table 6.1.1/1: Observations of sub-lethal effects and normal behaviour in the test vessels

Geometric mean measured concentration of the test item (mg/L)	Effect*	Number of fish effected at observation time (hours)
		3	24	48	72	96
0.999	(2.7) (2.6) (2.5) (2.1)	0 7 0 0	0 0 7 0	7 0 7 0	7 0 0 7	7 0 0 7
Control	(1)	7	7	7	7	7

*) The numbers in brackets correspond to the following observations: (1) = Normal behaviour (2.1) = Lethargy (2.5) = Missing escape reflex (2.6) = Quiescence (2.7) = Slowed down and unregularly breathing

Table 6.1.1/2: Cumulative mortality (%) in the test vessels

Geometric mean measured concentration of the test item (mg/L)	Cumulative mortality (%) after different exposure periods (hours)
	3	24	48	72	96
0.999	0	0	0	0	0
Control	0	0	0	0	0

Table 6.1.1/3: Measured concentrations and percent of the initial concentrations of the test item

Sampling date

0 hours Fresh medium

24 hours Old medium

24 hours Fresh medium

48 hours Old medium

48 hours Fresh medium

72 hours Old medium

72 hours Fresh medium

96 hours Old medium

Geometric mean measured concentration (mg/L)

Nominal concentration (mg/L)

Meas. conc. (mg/L)

Meas. conc. (mg/L)

%

Meas. conc. (mg/L)

Meas. conc. (mg/L)

%

Meas. conc. (mg/L)

Meas. conc. (mg/L)

%

Meas. conc. (mg/L)

Meas. conc. (mg/L)

%

1.50

1.03

0.693

67

1.15

0.801

70

1.38

0.968

70

1.20

0.940

78

0.999

Control

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

Meas. conc. = Measured concentration of the test item, enrichment and dilution factor taken into account.

% = Percent of initially measured concentration of the test item.

LOQ = Limit of Quantification (2 µg/L test item)

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Overall remarks”.

Conclusions:

The test resulted in an LC50 (96 h) of > 0.999 mg/L (geom. mean measured), indicating that the substance does not have lethal effects on zebrafish up to the threshold concentration of the saturated solution (OECD 203, D. rerio).

Executive summary:

The acute toxicity of the test substance to fish (zebrafish) was determined according to the principles of the OECD Guideline 203 (1992) and EU Method C.1 (2008), with GLP compliance. An acute toxicity test with the nominal threshold concentration of 1.50 mg/L (corresponding to a geometric mean measured concentration of 0.999 mg test item/L) was conducted under semi-static conditions with daily renewal of the test media. Duration of the test was 96 hours. Seven test organisms were exposed to the threshold loading and the control. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The concentrations of the test substance were analytically verified via GC-MS at the start (0, 24, 48, 72 h) and at the end (24, 48, 72, 96 h) of the exposure intervals in the tested concentration level and the control. The measured concentrations of the two constituents of the test substance at the end of each exposure interval were in the range of 67 to 78% of the initially measured concentrations. Therefore, all effect values given are based on the geometric mean measured test item concentration. All validity criteria of the test guideline were met.

After 96 h, sub-lethal effects were observed but no mortality was recorded. Therefore, the LC50 (96 h) corresponds to > 0.999 mg test item/L (geom. mean measured concentration).

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the structural similarity between the source and the target substances (stereoisomers) and comparable properties related to the target endpoints.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Refer to the Test material section of the source and target records.

3. ANALOGUE APPROACH JUSTIFICATION
See attached document in Iuclid section 13

4. DATA MATRIX
See attached document in Iuclid section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

Read-Across justification document

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.999 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Validity criteria fulfilled:

not applicable

Conclusions:

According to the experimental study performed on the source substance, the 96h-LC50 (Danio rerio) of the target substance is determined to be greater than 0.999 mg/L, the threshold concentration of the saturated solution.

Executive summary:

No experimental study is available on the target substance to assess the short-term toxicity of the registered substance to fish. Therefore, good quality data for a related source substance (Reaction Mass of 1-[(1R*,6S*)-2,2,6-Trimethylcyclohexyl]-3-hexanol and 1-[(1S*,6S*)-2,2,6-Trimethylcyclohexyl]-3-hexanol (EC#947-716-8)) have been read-across for this endpoint. The target and source substances are structurally related, in that both are reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The source substance is a reaction-mass of both cis (major) and trans isomers, with both 3R and 3S hydroxyl group (i.e. 4 diastereoisomers), whereas the target substance is a reaction-mass of two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group. The cis form (unknown absolute configuration, i.e. 1 or 2 possible isomers) being considered as an impurity. Thus, two isomers are common between the source and target substances, the other two being diastereoisomers. 

The acute toxicity to fish (Danio rerio), performed on the source substance, was conducted according to OECD Test Guideline 203 and EU Method C.1 with GLP statement. An acute toxicity test with the nominal threshold concentration of 1.50 mg/L (corresponding to a geometric mean measured concentration of 0.999 mg test item/L) was conducted under semi-static conditions with daily renewal of the test media. Duration of the test was 96 hours. Seven test organisms were exposed to the threshold loading and the control. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The concentrations of the test substance were analytically verified via GC-MS at the start (0, 24, 48, 72 h) and at the end (24, 48, 72, 96 h) of the exposure intervals in the tested concentration level and the control. The measured concentrations of the two constituents of the test substance at the end of each exposure interval were in the range of 67 to 78% of the initially measured concentrations. Therefore, all effect values given are based on the geometric mean measured test item concentration. All validity criteria of the test guideline were met.

After 96 h, sub-lethal effects were observed but no mortality was recorded. Therefore, the LC50 (96 h) corresponds to > 0.999 mg test item/L (geom. mean measured concentration), the threshold concentration of the saturated solution.

 Therefore, based on the considerations above, it can be concluded that the result of the acute fish study conducted with the source substance is likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 9.1.4 of the REACH regulation.

Description of key information

Read-across, OECD Guideline 203, EU Method C1, GLP, key study, validity 1:

No acute lethal effects were observed on Danio rerio up to the solubility limit of the test substance.

Additional information

To assess the short-term toxicity of the registered (target) substance to fish, one experimental study is available on a source substance.

This valid study was performed according to OECD Guideline 203 and EU Method C.1 in compliance with GLP on the source substance, Reaction Mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol (EC 947-716-8). The target and source substances are structurally related, in that both are reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The source substance is a reaction-mass of both cis (major) and trans isomers, with both 3R and 3S hydroxyl group (i.e. 4 diastereoisomers), whereas the target substance is a reaction-mass of two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group. The cis form (unknown absolute configuration, i.e. 1 or 2 possible isomers) being considered as an impurity. Thus, two isomers are common between the source and target substances, the other two being diastereoisomers. 

An acute toxicity test with the nominal threshold concentration of 1.50 mg/L (corresponding to a geometric mean measured concentration of 0.999 mg test item/L) was conducted under semi-static conditions with daily renewal of the test media. Duration of the test was 96 hours. Seven test organisms were exposed to the threshold loading and the control. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The concentrations of the test substance were analytically verified via GC-MS at the start (0, 24, 48, 72 h) and at the end (24, 48, 72, 96 h) of the exposure intervals in the tested concentration level and the control. The measured concentrations of the two constituents of the test substance at the end of each exposure interval were in the range of 67 to 78% of the initially measured concentrations. Therefore, all effect values given are based on the geometric mean measured test item concentration. All validity criteria of the test guideline were met.

After 96 h, sub-lethal effects were observed but no mortality was recorded. Therefore, the LC50 (96 h) corresponds to > 0.999 mg test item/L (geom. mean measured concentration), the threshold concentration of the saturated solution.

This source substance is considered adequate for read-across purposes based on structural similarity (stereoisomers), on similar basic physico-chemical properties and similar environmental profile between the registered substance and the analogue material used (see IUCLID section 13 for justification).