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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2014-11-18 to 2014-11-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD Guideline No. 202. Semi-stable emulsions may have been formed in the test solutions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on June 03-05, 2013 / signed on November 05, 2013
Specific details on test material used for the study:
- Water solubility in pure water: isomer 1 = 125 µg/L; isomer 2 = 285 µg/L
- Water solubility in M4 medium: isomer 1 = 115 µg/L; isomer 2 = 261 µg/L
(determined at Dr. U.NOACK-LABORATORIEN, Noack Lab-ID: 131029FG/CWE15836)
Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via GC-MS at the start (0 and 24 h) and at the end of both exposure intervals (24 and 48 h).
At the start of the exposure intervals (0 and 24 h), sampling was carried out after preparation of the test concentrations.
At the end of the exposure intervals (24 and 48 h), samples were taken directly from the test vessels.
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Quality criteria for the analytical monitoring:
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations. The effect levels have to be given based on the geometric mean measured concentrations, if the initially measured concentrations differ more than 20% from the nominal concentrations or a decrease of more than 20% from the initially measured concentration is observed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (1.00 mg/L of the test item were weighed out) was prepared with dilution water one day before the start of both exposure intervals (at -24 and 0 h). The stock solution was stirred with approximately 1100 rpm for 24 h at 25 ± 2 °C.
- Application: Per replicate, 20 g test solution were weighed out into the test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by a pipette.
- Controls: Dilution water without test item tested under the same conditions as the test groups.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), D-14195 Berlin, Germany.
- Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.
- Age at study initiation: 2 to 24 h old daphnids from a healthy stock were used for the study. They were obtained by removing the juvenile daphnids from the culture vessels latest 22 h before the start of the exposure. The juveniles born within this period of max. 22 h preceding the exposure were used for the test 2 hours after separation from the culture vessels. No first brood progeny was used for the test.
- Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2°C, in an incubator, 16 h illumination, light intensity of max. 20 µE/m2/s
- Culture feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL. The algae are cultured at the test facility.
- Origin of the food algae: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany.
- Feeding during test: The daphnids were not fed during the study.

ACCLIMATION
- Acclimation period: At least 2 h in dilution water
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
260 mg/L as CaCo3
Test temperature:
20.5-21.0 °C
pH:
0 h: 7.72
24 h: 7.78
Dissolved oxygen:
0 h: 8.87 mg/L
24 h: 8.77 mg/L
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 31.3, 62.5, 125, 250, 500 and 1000 µg/L.
Corresponding geometric mean measured concentrations: 24.1, 45.9, 94.4, 212, 378 and 863 µg/L.
Details on test conditions:
TEST METHOD:
A semi-static test design with a daily renewal of the test solutions in glass flasks sealed with screw caps (made from polypropylene) under diffuse light conditions was chosen to reduce losses of the test item concentrations by evaporation.

TEST SYSTEM
- Test vessels/volume: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm, ca. 130 mL) with screw caps (made from polypropylene) were used. They were filled up with the test solutions having nearly no headspace to reduce contact with air and losses of the test item concentrations by evaporation.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 5 (control and test material groups)
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Renewal of the test solutions: The test solutions were renewed after 24 h. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by a pipette.

TEST MEDIUM / WATER PARAMETERS
- Dilution water: ISO Test water, according to OECD 202
- Culture medium: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.
- Alkalinity: 260 mg CaCO3/L
- Conductivity: 647 µs

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/8 h dark cycle
- Light intensity: Diffuse light; light intensity of max. 20 µE/m2/s

EFFECT PARAMETERS MEASURED:
Biological parameters:
- Immobilisation and other observations: Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other relevant observations were not made.

Water Quality Parameters:
Dilution water: Prior to the start of the exposure (0 h) and the water renewal (24 h), the water quality parameters (i.e. pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.
Test media: At the start of the exposure intervals (0 and 24 h), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen concentration) were measured in one additional replicate per concentration level and control. At the end of the exposure intervals (24 and 48 h), the water parameters of the old media were measured in all replicates per concentration level and control.
Temperature: The incubator temperature was recorded throughout the period of the test.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Factor 2
- Range finding study: Test concentrations were selected based on the results of the most recent of a series of four non GLP preliminary range finding tests and the limit of water solubility of the test item in the dilution water, which was observed at approximately 1.00 mg/L. In the most recent preliminary range finding test performed under semi-static conditions with three concentrations of the test item of 10, 100 and 1000 µg/L and two replicates per concentration level with 10 daphnids each were tested in a closed system (glass flask completely filled up with the test solutions having no headspace). The percentage immobility was determined in all concentrations and control groups after 24 and 48 h of exposure.
- Results used to determine the conditions for the definitive study: After 48 h of exposure the total rate of immobilisation of daphnia was 85%, 20% and 5% at 1000, 100 and 10 µg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
522 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: > solubility limit
Duration:
48 h
Dose descriptor:
other: The highest tested concentration without observed effect
Effect conc.:
212 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Biological data:
Immobilization of daphnids for the definitive test was 0, 0, 0, 0, 0, 15 and 50 % (24 h); 0, 0, 0, 0, 0, 25 and 85 % (48 h) at 31.3, 62.5, 125, 250, 500 and 1000 µg/L, respectively.
- Measured Exposure Concentrations during the Definitive Test:
The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 81 to 98 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 56 to 88 % of the nominal values. The geometric mean measured concentrations of the test item were calculated to be: 24.1, 45.9, 94.4, 212, 378 and 863 µg/L. Note that the solubility limit is measured at 376 µg/L in M4 medium according to the OECD Guideline 105 study, performed with a slow-stirring method. Therefore, toxic effects were observed at concentrations greater than the solubility limit.
- Appearance of test solutions: Semi-stable emulsions may have been formed in the test solutions. These emulsions, particularly at concentrations ≤ 1 mg/L, are generally invisible, that is why no any undissolved particles were observed.
- Water Quality Parameters:
The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.
Results with reference substance (positive control):
The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.57 mg/L (CI 1.53 - 1.60 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.
Reported statistics and error estimates:
EC values and statistical analysis:
The EC50-values after 24 and 48 h were calculated by sigmoidal dose-response regression. The 95 % confidence limits were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5. The concentration-effect relationships are shown graphically. The EC50 -value for the reference item and its confidence limits were calculated accordingly.

Table 6.1.3/1: Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

 

Nominal

test item

concentration

 

[µg/L]

Geometric mean

measured

test item

concentration

[µg/L]

IMMOBILISATION [%]

24 h

48 h

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

1000

863

60

60

40

40

50

80

80

100

80

85

 500

378

 0

 0

60

 0

15

20

 0

 60

20

25

 250

212

 0

 0

 0

 0

 0

 0

 0

   0

 0

 0

 125

 94.4

 0

 0

 0

 0

 0

 0

 0

   0

 0

 0

 62.5

 45.9

 0

 0

 0

 0

 0

 0

 0

   0

 0

 0

 31.3

 24.1

 0

 0

 0

 0

 0

 0

 0

   0

 0

 0

Control

 0

 0

 0

 0

 0

 0

 0

   0

 0

 0

n = 20, divided into 4 replicates with 5 daphnids each

MV   = mean value

 

Table 6.1.3/2: Measured Concentrations of the Test Item during the Definitive Test (based on the determination of 3 Isomers)

 

Sampling date

2014-11-25

0 hours

Start of the

exposure interval

2014-11-26

24 hours

End of the

exposure interval

2014-11-26

24 hours

Start of the

exposure interval

2014-11-27

48 hours

End of the

exposure interval

Geometric mean measured concentration

Start of analysis

2014-11-25

2014-11-26

2014-11-26

2014-11-27

Nominal

test item

concentration

[µg/L]

Test item

Meas.

conc.

[µg/L]1)

%

Meas.

conc.

[µg/L]

%

Meas.

conc.

[µg/L]

%

Meas.

conc.

[µg/L]

%

Meas.

conc.

[µg/L]

%

1000

949

95

858

86

981

98

694

69

863

86

500

413

83

371

74

458

92

291

58

378

76

250

230

92

220

88

219

88

181

72

212

85

125

101

81

91.0

73

1232)

98

70.1

56

94.4

76

62.5

51.9

83

46.1

74

51.1

82

36.4

58

45.9

73

31.3

30.82)

98

19.8

63

26.9

86

20.4

65

24.1

77

Control

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

Meas. conc.= measured concentration of the test item, single injection, dilution factors taken into account

% = percent of the nominal concentration of the test item

LOQ = limit of quantification (10 µg test item/L)

1) Analysed on 2014-11-26

2) Reanalysed on 2014-11-27, mean value of two replicates

Table 6.1.3/3: Distribution of Isomers in the Test Item Concentrations during the Definitive Test (sum of isomers defined as 100 %)

 

Sampling date

2014-11-25

0 hours

Start of the

exposure interval

2014-11-26

24 hours

End of the

exposure interval

2014-11-26

24 hours

Start of the

exposure interval

2014-11-27

48 hours

End of the

exposure interval

Start of analysis

2014-11-25

2014-11-26

2014-11-26

2014-11-27

Nominal

test item

concentration

[µg/L]

Distribution of Isomers of test item

Isomer

1

[%]

Isomer

2

[%]

Sum

 

[%]

Isomer

1

[%]

Isomer

2

[%]

Sum

 

[%]

Isomer

1

[%]

Isomer

2

[%]

Sum

 

[%]

Isomer

1

[%]

Isomer

2

[%]

Sum

 

[%]

1000

49.0

51.0

100

49.9

50.1

100

51.2

48.8

100

50.3

49.7

100

500

49.0

51.0

100

50.6

49.4

100

51.0

49.0

100

50.4

49.6

100

250

49.6

50.4

100

50.8

49.3

100

49.8

50.2

100

50.6

49.4

100

125

49.8

50.2

100

51.5

48.5

100

51.6

48.4

100

50.6

49.4

100

62.5

49.2

50.8

100

49.0

51.0

100

51.1

48.9

100

50.4

49.6

100

31.3

49.4

50.6

100

50.7

49.3

100

51.0

49.1

100

50.5

49.5

100

%= percentage of the peak area of each isomer, based on the sum of peak areas defined as 100 %

 

 

Table 6.1.3/4: Calculated Concentrations of both Isomers of the Test Item during the Definitive Test

Sampling date

2014-11-25

0 hours

Start of the

exposure interval

2014-11-26

24 hours

End of the

exposure interval

2014-11-26

24 hours

Start of the

exposure interval

2014-11-27

48 hours

End of the

exposure interval

Start of analysis

2014-11-25

2014-11-26

2014-11-26

2014-11-27

Nominal

test item

concentration

[µg/L]

Calculated Concentration for each Isomer of the Test Item*

 

Isomer

1

[µg/L]

Isomer

2

[µg/L]

Sum

 

[µg/L]

Isomer

1

[µg/L]

Isomer

2

[µg/L]

Sum

 

[µg/L]

Isomer

1

[µg/L]

Isomer

2

[µg/L]

Sum

 

[µg/L]

Isomer

1

[µg/L]

Isomer

2

[µg/L]

Sum

 

[µg/L]

1000

465

484

949

428

430

858

502

479

981

349

345

694

500

202

211

413

188

183

371

234

224

458

147

144

291

250

114

116

230

112

108

220

109

110

219

91.6

89.4

181

125

50.3

50.7

101

46.9

44.1

91.0

63.5

59.5

123

35.5

34.6

70.1

62.5

25.5

26.4

51.9

22.6

23.5

46.1

26.1

25.0

51.1

18.3

18.1

36.4

31.3

15.2

15.6

30.8

10.0

9.76

19.8

13.7

13.2

26.9

10.3

10.1

20.4

*= calculated from the determined test item concentration and the percentage of isomer distribution in the test concentration

 

Validity criteria:

- In the control group, no daphnids were immobilized or showed any signs of disease or stress, e.g. discoloration or unusual behaviour such as trapping on the surface of the water, during the 48 h test period (required: not more than 10 per cent of the daphnids in the control).

- The dissolved O2 concentration at the end of the exposure intervals was ≥ 8.01 mg/L (required: ≥ 3 mg/L at the test end of the exposure intervals) in the test vessels of all tested concentration levels and the control.

Validity criteria fulfilled:
yes
Conclusions:
No acute effects were observed after 48 hours on Daphnia magna up to the solubility limit of the test substance.
Executive summary:

Study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the test substance to Daphnia magna, under semi-static conditions.

Test item was exposed to daphnids at the concentrations of 31.3, 62.5, 125, 250, 500 and 1000 µg/L for 48 hours. The no-treatment control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 20.5-21.0 °C. The tested solutions were clear throughout the exposure period. Before definitive test, a 48-hour semi-static range-finding test was conducted and the percent immobilization was 85%, 20% and 5% at 1000, 100 and 10 µg/L, respectively.

 

The concentrations of the test item were analytically verified via GC-MS at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h) in all concentration levels and the control. The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 81 to 98 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 56 to 88 % of the nominal values. The geometric mean measured concentrations of the test item were calculated to be 24.1, 45.9, 94.4, 212, 378 and 863 µg/L. According to the water solubility study, performed in compliance with the OECD Guideline 105 with a slow-stirring method, the solubility limit measured in Daphnia medium was 376 µg/L. Therefore, semi-stable emulsions may have been formed in the test solutions. These emulsions, particularly at concentrations lower than 1 mg/L, are generally invisible, that is why no any undissolved particles were observed.

 

Immobilization of daphnids for the definitive test was 0, 0, 0, 0, 0, 15 and 50 % (24 h); 0, 0, 0, 0, 0, 25 and 85 % (48 h) at geometric mean measured concentrations of 24.1, 45.9, 94.4, 212, 378 and 863 µg/L, respectively. Therefore, toxic effects were observed only at concentrations greater than the solubility limit. The 48h-EC50 value was determined to be 522 µg/L (Cl: 457 – 607 µg/L), which is greater than the solubility limit. The EC50 -value of the reference item, potassium dichromate at 1.57 mg/L (CI 1.53 - 1.60 mg/L) after 24 h was within the prescribed concentration range of 0.6 - 2.1 mg/L.

 

In conclusion, no acute effects were observed on Daphnia magna up to the solubility limit of the test substance.

Endpoint:
short-term toxicity to aquatic invertebrates
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:
48 h
Dose descriptor:
EC50
Effect conc.:
522 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: > solubility limit
Duration:
48 h
Dose descriptor:
other: The highest tested concentration without observed effect
Effect conc.:
212 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Validity criteria fulfilled:
yes
Conclusions:
According to the experimental study performed on the source substance, no acute effects were observed after 48 hours on Daphnia magna up to the solubility limit of the test substance.
Executive summary:

No experimental study is available on the target substance to assess the short-term toxicity of the registered substance to aquatic invertebrates. Therefore, good quality data for a related source substance (Reaction mass of (3R*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and (3S*)-1-[(1R*,6S*)-2,2,6-Trimethylcyclohexyl]hexan-3-ol (EC# 942-425-2)) have been read-across for this endpoint. The target and the source substance are structurally related, in that both are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group, of the target/registered substance are constituents of the source substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantionmers). 


The acute toxicity to Daphnia magna, performed on the source substance, was conducted according to OECD Test Guideline 202 with GLP statement, under semi-static conditions. Test item was exposed to daphnids at the concentrations of 31.3, 62.5, 125, 250, 500 and 1000 µg/L for 48 hours. The no-treatment control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 20.5-21.0 °C. The tested solutions were clear throughout the exposure period. Before definitive test, a 48-hour semi-static range-finding test was conducted and the percent immobilization was 85%, 20% and 5% at 1000, 100 and 10 µg/L, respectively.


The concentrations of the test item were analytically verified via GC-MS at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h) in all concentration levels and the control. The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 81 to 98 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 56 to 88 % of the nominal values. The geometric mean measured concentrations of the test item were calculated to be 24.1, 45.9, 94.4, 212, 378 and 863 µg/L. According to the water solubility study, performed in compliance with the OECD Guideline 105 with a slow-stirring method, the solubility limit measured in Daphnia medium was 376 µg/L. Therefore, semi-stable emulsions may have been formed in the test solutions. These emulsions, particularly at concentrations lower than 1 mg/L, are generally invisible, that is why no any undissolved particles were observed.


Immobilization of daphnids for the definitive test was 0, 0, 0, 0, 0, 15 and 50 % (24 h); 0, 0, 0, 0, 0, 25 and 85 % (48 h) at geometric mean measured concentrations of 24.1, 45.9, 94.4, 212, 378 and 863 µg/L, respectively. Therefore, toxic effects were observed only at concentrations greater than the solubility limit. The 48h-EC50 value was determined to be 522 µg/L (Cl: 457 – 607 µg/L), which is greater than the solubility limit. The EC50 -value of the reference item, potassium dichromate at 1.57 mg/L (CI 1.53 - 1.60 mg/L) after 24 h was within the prescribed concentration range of 0.6 - 2.1 mg/L.


In conclusion, no acute effects were observed on Daphnia magna up to the solubility limit of the test substance.


Therefore, based on the considerations above, it can be concluded that the result of the acute daphnia 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 VII, 9.1.4 of the REACH regulation.

Description of key information

Read-across, OECD Guideline 202, GLP, key study, validity 2:

No acute effects were observed on Daphnia magna up to the solubility limit of the test substance.

Key value for chemical safety assessment

Additional information

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


This valid GLP study, assessed as a key study, was performed on the source substance, Reaction mass of (3R*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and (3S*)-1-[(1R*,6S*)-2,2,6-Trimethylcyclohexyl]hexan-3-ol (EC# 942-425-2), according to OECD Guideline 202. The target and the source substance are structurally related, in that both are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group, of the target/registered substance are constituents of the source substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantionmers). 


In this study, twenty Daphnia magna were exposed to each concentration level of the test substance (nominal concentrations: 31.3, 62.5, 125, 250, 500 and 1000 µg/L) and the control, in closed system (sealed glass flasks) under semi-static conditions, over a period of 48 hours. The concentrations of the test substance were analytically verified via GC-MS at the start of the exposure intervals (0 and 24h) and at the end of the exposure intervals (24 and 48h) in all concentration levels and the control. The measured concentrations of the test substance at the start of the exposure intervals were in the range of 81 to 98 % of the nominal values. At the end of the exposure intervals, the measured concentrations of the test substance were in the range of 56 to 88 % of the nominal values. Therefore, the geometric mean measured concentrations of the test substance were calculated to be: 24.1, 45.9, 94.4, 212, 378 and 863 µg/L. According to the water solubility study, performed on the read-across substance in compliance with the OECD Guideline 105 with a slow-stirring method, the solubility limit measured in Daphnia medium was 376 µg/L (as the sum of two isomers). Therefore, semi-stable emulsions may have been formed in the test solutions. These emulsions, particularly at concentrations lower than 1 mg/L, are generally invisible, that is why no any undissolved particles were observed. Immobilization of daphnids for the definitive test was 0, 0, 0, 0, 0, 15 and 50 % (24 h); 0, 0, 0, 0, 0, 25 and 85 % (48 h) at geometric mean measured concentrations of 24.1, 45.9, 94.4, 212, 378 and 863 µg/L, respectively. According to this result, toxic effects were observed only at concentrations greater than the solubility limit. The first effects (25% immobilisation at 48 h) were observed at a geometric mean concentration of 378 µg/L, just above the solubility limit. The 48h-EC50 value was determined to be 522 µg/L(Cl: 457 – 607 µg/L), which is significantly greater than the solubility limit. In conclusion, no acute effects were observed on Daphnia magna up to the solubility limit of the test substance.


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).