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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January, 08, 2019 to February, 20, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Remarks:
HPLC-DAD
Details on sampling:
Preparation of samples for definitive test on 21.01.2019 (0 hour) and 24.01.2019 (72 hour):
Control (T1, T2), Treatment sample, T3 were analyzed as such and T5, T7 samples were further diluted as below mentioned:

Sample ID Volume pipetted µL Volume diluted (mL) using acetonitrile
T5 (7.0 mg/L) 3500 5
T7 (49.3 mg/L) 1000 10
Vehicle:
yes
Remarks:
solvent (acetone)
Details on test solutions:
Vehicle Selection: An amount of 10.487 mg of the test was weighed, dissolved with 10µl of Acetone and made upto 10.0 ml of OECD media in a standard volumetric flask as a stock. From stock, 9.54ml was taken and transferred to 100 ml volumetric flask and made upto the mark with OECD media and found to be soluble hence, acetone was used as a vehicle.
Justification for Selection of Vehicle: Since test item was insoluble in water as detailed in Safety Data Sheet given by the Sponsor. Based on the information provided by sponsor, it was determined that the test item was not soluble in OECD medium. Hence the test item was treated initially with 0.1ml/L Acetone to aid the test item to get soluble in OECD medium.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source: Bharathiar University and sub cultured in house
Initial biomass concentration: 1 x 10^4 cells/mL.
Photoperiod: Continuous light
Test type:
static
Water media type:
other: OECD medium
Total exposure duration:
72 h
Test temperature:
Range finding test: 22.2°C – 22.6°C
Definitive test: 22.3°C – 22.6°C
pH:
pH was recorded at 0 hour (bulk samples) and 72 hours (pooled samples) of each test concentrations and control.
Range finding test: 8.0 – 8.2
Definitive test: 8.0-8.1
Nominal and measured concentrations:
Test concentrations of 1.0, 2.7, 7.0, 18.6 and 49.3 mg/L of Safranal with a spacing factor of 2.65 along with a control and solvent control without test item were selected for definitive test.
Details on test conditions:
Light (Lux): Range finding test: 4627 - 4634 / Definitive test: 4627-4656
Photoperiod: Continuous light
RPM: Range finding test: 124 to 130 / Definitive test: 124 to 130
Accommodation: 250 ml conical flask with cotton plug
Medium: OECD medium
Volume of medium: 100 mL

1- Range Finding Test
Control(s): 1- OECD medium / 2- Solvent control (0.1ml/L of Acetone)
Treatment: 1, 10, 25, 50 and 100 mg/L of Concentrations
Replication: 3 replicate for each test concentration and 6 replicate for control and solvent control.

2- Definitive Test
Control(s): 1- OECD medium / 2- Solvent control (0.1ml/L of Acetone)
Treatment: 1.0, 2.7, 7.0, 18.6 and 49.3 mg/L concentration with geometric series separation factor 2.65.
Replication: Control and solvent control 6 replicates, Each test concentration 3 replicate.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.88 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Observations: At the end of 72 hour observation, 0.78%, 10.56%, 18.39%, 44.21% and 94.53% growth inhibition and 3.43%, 35.77%, 53.79%, 84.98% and 99.57% yield inhibition was observed in the test concentrations of 1.0, 2.7, 7.0, 18.6 and 49.3 mg/L respectively.
- Dose Verification: During the definitive test, control, solvent control, 1.0, 7.0 and 49.3 mg/L (Low, Mid and High) concentration of sample 0 hour and 72 hour sample were sent to the Analytical chemistry at VBS for dose verification analysis. The test item concentrations, control and solvent control was analyzed only for definitive test. The test concentration was maintained within 80-120% of the nominal concentration, throughout the exposure period, the results were based on the nominal concentrations.
- NOEC and LOEC: These are reported from growth rate as less than 1 mg/L and 1 mg/L, respectively and for yield are less than 1 mg/L and 1 mg/L, respectively. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the EC10 is presented in the above table.
Reported statistics and error estimates:
The effective concentrations based on growth rate and yield were calculated from the 72 hour algal cell count. The EC50/20/10 values based on the inhibition of growth rate and yield were calculated using probit analysis. The 95% confidence limits were calculated from the probit analysis results.
The NOEC and LOEC were calculated using Kruskal-Wallis multiple comparison test with one-way analysis of variance with significance at alpha value 0.05. The data was accepted for homogeneity of variance and normality for ANOVA. All the statistical analysis was done using NCSS 12.0.4.
Validity criteria fulfilled:
yes
Conclusions:
The results of the study shows that Test item at tested concentrations has inhibitory effects on the growth rate and yield of Pseudokirchneriella subcapitata during the 72 hour exposure period. The validity criteria according to the OECD guideline 201 have been met in this study. Based on nominal concentrations, the 72 hour ErC50 is 15.50 mg/L and the 72 hour EyC50 is 5.36 mg/L. The corresponding EC10 values are 3.88 and 1.33 mg/L. The NOEC and LOEC for growth rate are less than 1 mg/L and 1 mg/L, respectively and for yield are less than 1 mg/L and 1 mg/L, respectively.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for risk assessment classification purposes, which were determined in this study to be 15.5 mg/L and 3.88 mg/L respectively.
Executive summary:

The objective of this study was to assess the Growth Inhibition Study of Safranal in Freshwater Alga (Pseudokirchneriella subcapitata) followed by the period of 72 hours observation. The study was performed in compliance with following regulatory guideline: OECD Guidelines for the Testing of Chemicals, Section.2. Number 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” Adopted on 23 March 2006 Annexure 5 corrected: 28 July 2011. The control and treatment flasks were inoculated with Pseudokirchneriella subcapitata precultured for three days and tested at an initial cell density of 1 × 104 cells per mL and incubated under test condition for 72 hours. The cells were counted using an Improved Neubaur’s Haemocytometer under illumination of the microscope at 24, 48 and 72 hour after inoculation.

A range finding test was conducted with Test item at the concentrations of 1, 10, 25, 50 and 100.0 mg/L along with control and solvent control. Each test concentrations consists of three replicate whereas six replicates was maintained for control and solvent control group. No significant changes in the appearance of algae cells were observed in control and in the test concentrations during 72 hours test period. At the end of 72 hour observation, 2.29%, 17.43%, 51.01%, 91.02% and 94.86% growth inhibition and 9.12%, 51.76%, 88.94%, 99.14% and 99.57% yield inhibition was observed in the test concentrations of 1, 10, 25, 50 and 100 mg/L respectively.

Based on the results of range finding test, a definitive test was conducted with the test concentrations of 1.0, 2.7, 7.0, 18.6 and 49.3 mg/L (geometric factor of 2.65) along with control and solvent control. Each test concentrations three replicate were used and six replicates was maintained for control and solvent control group. No significant changes in the appearance of algae cells were observed in controls and in the test concentrations during 72 hours test period. At the end of 72 hour observation, 0.78%, 10.56%, 18.39%, 44.21% and 94.53% growth inhibition and 3.43%, 35.77%, 53.79%, 84.98% and 99.57% yield inhibition was observed in the test concentrations of 1.0, 2.7, 7.0, 18.6 and 49.3 mg/L respectively.

During the test period, all the flasks were incubated in a shaker incubator under controlled conditions. The temperature during the test period maintained between for range finding test 22.4°C to 22.6°C and for definitive test 22.3°C – 22.6°C. In light intensity for range finding test and definitive test ranged between 4627 to 4634 lux and 4627 to 4656 lux average agitation speed was about 127 RPM for range finding test and for definitive test 128 RPM. The pH of control, solvent control and test concentrations at the beginning of the test and at test termination was 8.0 to 8.2 for range finding test and for definitive test 8.0 to 8.1.

The effective concentrations based on growth rate and yield were calculated from the 72 hour algal cell count. The EC50/20/10 values based on the inhibition of growth rate and yield were calculated using probit analysis. The 95% confidence limits were calculated from the probit analysis results. The NOEC and LOEC were calculated using Kruskal-Wallis multiple comparison test with oneway analysis of variance with significance at alpha value 0.05. The data was accepted for homogeneity of variance and normality for ANOVA. All the statistical analysis was done using NCSS 12.0.4.

Based on the results of definitive test, the nominal concentrations, the 72 hour ErC50 is 15.50 mg/L and the 72 hour EyC50 is 5.36 mg/L. The NOEC and LOEC for growth rate are less than 1 mg/L and 1 mg/L, respectively and for yield are less than less than 1 mg/L and 1 mg/L, respectively.

ECx, NOEC and LOEC based on Inhibition of Growth Rate and Yield (mg/L):

Response variable based on % inhibition (0-72 h)  Effective concentration  95% confidence limits 
Lower limit Upper Limit
Growth rate ErC10 3.88 2.94 4.82
ErC20 6.24 5.04 7.44
ErC50 15.5 13.05 17.95
Yield EyC10 1.33 1 1.66
EyC20 2.14 1.69 2.59
EyC50 5.36 4.54 6.18
Growth rate NOEC Less than 1 mg/L
LOEC 1 mg/L
Yield NOEC Less than 1 mg/L
LOEC 1 mg/L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From October 7, 2011 to October 28, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is used for read-across to 2,6,6-trimethylcyclohexa-1,3-dienecarbaldehyde.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Nominal and measured concentrations:
Nominal: 2.2, 4.4, 8.8, 18, 35, and 70 mg/L
Mean Measured Concentrations (72h): 1.1, 1.6, 5.9, 12 and 28 mg/L
Mean Measured Concentrations corrected for algal uptake (72h): 1.3, 2.5, 6.8, 15 and 32 mg/L
Mean Measured Concentrations (96h):0.79, 1.4, 4.4, 11 and 28 mg/L
Mean Measured Concentrations corrected for algal uptake (72h): 0.99, 2.5, 5.1, 14 and 34 mg/L
Details on test conditions:
As the test item is a volatile substance, the test was performed using Erlenmeyer flasks completely filled with test medium and tightly sealed with glass stoppers to avoid losses of test item (closed system).

In order to evaluate the influence of the presence of light and algae on the stability of the test item, additional flasks containing the test medium of 18 mg/L (also used for the toxicity testing) were set up in a closed system. Three different incubation conditions, “Light with algae”, “Light without algae”, and “Darkness without algae”, were applied.
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 19 - 25
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 16 - 20
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 12-14
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 9.8-13
Details on results:
Effects based on yield were also reported (see attached full study report). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are present in the above "effects concentration" table.

The measured concentrations at the start of the test were 84 to 88% of the nominal values. Thus, the correct dosage of the test item at test start could be verified. Over the test period of 96 hours the test item concentrations in the test media decreased. In a parallel stability study the main loss of test substance in the test water was attributed to adsorption to the growing algal biomass which accounted for approximately 13% decrease of the starting concentration over the period of 48 hours, 27% over 72 hours, and, 72% over 96 hours. This loss was considered in the calculation of the study endpoints, as the algal cells were, in effect, exposed to this complimentary fraction of test substance i.e. the biological response results are based upon mean measured concentrations corrected for algal uptake. Losses due to aqueous photolysis were only in the order of 5%.

A recovery phase of 10 days followed the exposure period. In all treatments, the test item GR-50-0091 had only algistatic effects on Pseudokirchneriella subcapitata. The inhibitory effects after the exposure period of 96 hours were reversible and re-growth occurred in all treatments.
Validity criteria fulfilled:
yes
Conclusions:
In a guideline study, conducted according to GLP, Shisolia (GR-50-0091) was found to have a 72h ErC50 of 18 mg/L and a 96h ErC50 of 21 mg/L.

This experimental result is being used to read-across to the registration substance, 2,6,6-trimethylcyclohex-1,3-dienecarbaldehyde (see target record).
Executive summary:

The influence of the test item GR-50-0091 on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test according to the OECD Guideline 201 (2006), and the Commission Regulation (EC) No 761/2009, C.3, as well as according to the OPPTS Guideline No. 850.5400 (Public Draft, April 1996).

The nominal concentrations of the test item of 2.2, 4.4, 8.8, 18, 35, and 70 mg/L were tested in parallel with a control.

As the test item is a volatile substance, the test was performed using Erlenmeyer flasks completely filled with test medium and tightly sealed with glass stoppers to avoid losses of test item (closed system). Three replicates per treatment with nominal 5’00fds0 algal cells/mL were exposed to the test item during 96 hours under continuous lighting at 21 °C. The algal biomass in the samples was determined by fluorescence measurement. At the end of the test, the shape and size of the algal cells were visually inspected, and, no adverse effect was observed.

At the end of the 96 hour exposure period, the test item media were diluted at a ratio of 1.5 mL test medium/100mL reconstituted water and re-growth of the algal cells was determined over a 10-day period in order to observe potential algistatic and algicidal effects.

In order to evaluate the influence of the presence of light and algae on the stability of the test item, additional flasks containing the test medium of 18 mg/L (also used for the toxicity testing) were set up in a closed system. Three different incubation conditions, “Light with algae”, “Light without algae”, and “Darkness without algae”, were applied.

All samples were stored frozen, thawed prior to analysis and the algal cells separated from the aqueous phase by centrifugation. Analysis was performed by GC-FID following extraction of samples into an organic solvent.

At the start of the test, 84 to 88% of the nominal values were measured in the test media of the test item concentrations of 2.2, 4.4, 8.8, 18 and 35 mg/L. Thus, the correct dosage of the test item at test start could be verified. Over the test period of 96 hours the test item concentrations in the test media decreased. After 72 hours and 96 hours of test duration, test item concentrations had decreased to 5 - 78% and <LOQ - 69% (LOQ = 0.116 mg/L, limit of quantification) of the values measured at test start, respectively. Samples of the highest test item concentration (nominal 70 mg/L) were not analyzed since this concentration was above the EC100 determined in this test.

In the parallel stability study the main loss of test substance in the test water was attributed to the presence of algal cells which accounted for approximately 13% of decrease of the starting concentration over the period of 48 hours, 27% over 72 hours, and, 72% over 96 hours. This loss was considered in the calculation of the study endpoints, as the algal cells were, in effect, exposed to this complimentary fraction of test substance. This conversion factor is considered conservative for application to the overall calculations given that the profusion of algal cells was much greater at test concentrations below that of the concentration level at which the stability study was performed (18 mg/L). Losses due to aqueous photolysis were the order of 5%.

 Incubation Conditions

 

 0 Hours

48 Hours

72 Hours

 96 Hours

 Light + Algae

 Concn. (mg/L)

 15.9

12.6 

8.24 

2.02 

 Light + Algae

 % of Day 0

 100

79 

52 

13 

 Light without Algae

 Concn. (mg/L)

 15.9

14.6 

12.5 

13.5 

 Light without Algae

 % of Day 0

 100

92 

79 

85 

Darkness without Algae

 Concn. (mg/L)

 15.9

14.4 

12.0 

14.4 

Darkness without Algae

 % of Day 0

 100

91 

76 

91 

The 72- and 96-hours mean measured concentrations were as follows:

   Mean measured concentrations Mean measured concentrations   Mean measured concentrations  Mean measured concentrations
   72 hours  72 hours  96 hours  96 hours

Nominal concentrations

(mg/L)

Mean measured concentrations (mg/L)

Mean measured concentrations corrected

for algal uptake + (mg/L)

Mean measured concentrations (mg/L)

Mean measured concentrations corrected

for algal uptake + (mg/L)

 2.2*

1.1 

1.3 

0.79 

0.99 

 4.4*

1.6 

 2.5

1.4 

2.5 

 8.8*

5.9 

6.8 

4.4 

5.1 

 18

12 

15 

11 

14 

 35

28 

32 

28 

34 

* at this treatment the measured concentration at the end of the test was <LOQ; the½ LOQ (0.058 mg/L) was used as 96-hour-concentration to calculate the time-weighted means.

+ Mean measured or time-weighted concentrations calculated from sample injections and corrected for “adsorption factor” (13% for 48-hour values, 27% for 72-hour values, 72% for 96-hour values).

The biological results of the study were as follows (based on mean measured test item concentrations):

Effect concentration (mg/L)

 72 hours - Growth rate

  72 hours - Yield

 96 hours - Growth rate

 96 hours - Yield

EC50 (95% CI)

 Direct Analysis

 15

(14 -16)

7.2

(6.2 -8.3) 

17

(15 -19) 

8.9

(7.9 -10) 

EC50 (95% CI)

Corrected for Algal Uptake

 18

(16 -20)

8.5

(7.5 -9.6) 

21

(19 -25) 

11

(10 -13) 

EC20 (95% CI)

 Direct Analysis

 11

(10 -12)

3.6

(2.5 -4.5) 

12

(12 -13) 

4.5

(3.6 -5.3) 

EC20 (95% CI)

Corrected for Algal Uptake

 14

(12 -14)

4.2

(3.3 -5.0) 

16

(15 -17) 

5.5

(4.2 -6.6) 

EC10 (95% CI)

 Direct Analysis

 9.5

(8.0 -10)

2.5

(1.5 -3.3) 

10

(9.3 -11) 

3.2

(2.3 -3.9) 

EC10 (95% CI)

Corrected for Algal Uptake

 12

(9.8 -13)

2.9

(2.1 -3.6) 

13

(12 -14) 

3.8

(2.7 -4.8) 

95% CI: 95% confidence interval

 Effect concentration (mg/L)

 72 hours - Growth rate

 72 hours - Yield

 96 hours - Growth rate

 96 hours - Yield

 NOEC

 Direct Analysis

 1.6

1.1 

1.4 

1.4 

 NOEC

 Corrected for Algal Uptake

 2.5

1.3 

2.5 

2.5 

 LOEC

 Direct Analysis

 5.9

1.6 

4.4 

4.4 

 LOEC

 Corrected for Algal Uptake

 6.8

2.5 

5.1 

5.1 

A recovery phase of 10 days followed the exposure period. In all treatments, the test item GR-50-0091 had only algistatic effects on Pseudokirchneriella subcapitata. The inhibitory effects after the exposure period of 96 hours were reversible and re-growth occurred in all treatments.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
A detailed read-across justification in-line with the ECHA RAAF guidelines is provided as an attached document. In summary,

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
- The source and target substance have similar ecotoxicological properties as a result of structural similarity, the same expected mode of action for aquatic toxicity and similar hydrophobicity (as modelled by log Kow).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- see read-across justification for details

3. ANALOGUE APPROACH JUSTIFICATION
- The structures of the main constituents of the target and source substance are very similar. All structures contain an aldehyde group attached to a cyclohexyl ring with unsaturation either in the 2- or 3-position relative to the aldehyde group. As such, the three acute aquatic toxicity profilers in the OECD Toolbox assign the main constituent of the target substance (2,6,6-trimethylcyclohexa-1,3-dienecarbaldehyde) and the two main constituents of source substance (Shisolia) to the same mode of action classes: ECOSAR class = vinyl/allyl aldehyde; OASIS MOA = aldehyde; Verhaar class = 3, unspecific reactivity.
- There are no impurities or additives that are expected to affect the ecotoxicological properties of the source and target substance
- The measured log Kow values for the target and source substance are 2.7 and 2.4 respectively. These two values only vary by 0.3 log units and as such the strength of aquatic toxicity for the two substances is expected to be similar. This is supported by experimental acute fish data and estimated E(L)C50 values (see below).
- Acute fish toxicity data is available for the target and source substance. The determined LC50 values are respectively, 4.3mg/L and 9.2mg/L (see IUCLID endpoint 6.1.1 for details).
- ECOSAR estimates for the target and source substance are also similar. For the algal endpoint, the subject of this read-across justification, the ECOSAR vinyl/allyl aldehyde SAR estimates a 96h EC50 value of 22.7 mg/L for the source substance compared with a value of 15.9 mg/L for the target substance. These estimates are considered reliable given that the experimental data for the source substance (Shisolia, 96h ErC50 of 21 mg/L) is in close agreement with the ECOSAR prediction.

4. DATA MATRIX
See Table 2 in attached read-across justification document
Reason / purpose for cross-reference:
read-across source
Specific details on test material used for the study:
The source substance (trade name: Shisolia, EC 700-805-3) is a multi-constituent substance; Reaction mass of 1-vinylcyclohex-3-enecarbaldehyde (CAS 1049017-63-1, concentration range of 60-70%) and 4-vinylcyclohex-1-enecarbaldehyde (CAS 1049017-68-6, concentration range of 25-35%). These two components are closely related in structure to the mono-constituent of the registration substance, 2,6,6-trimethylcyclohexa-1,3-dienecarbaldehyde (tradename: Safranal) and have the same expected mode of action for aquatic toxicity. Comparison of physicochemical properties (i.e. log Kow), available aquatic toxicity fish data and ECOSAR estimates show Shisolia to be a good surrogate for read-across to Safranal P. For details see the Read-Across Justification document attached above.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: direct read-across
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
15 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: corrected for differences in log Kow
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12.9 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: corrected for differences in log Kow
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: direct read-across
Details on results:
The measured log Kow of the target substance (Safranal, log Kow 2.7) is slightly higher than that for the source substance (Shisolia, log Kow 2.4). Therefore Safranal is expected to be slightly more toxic. This is supported by the experimental fish LC50 values of 4.3 and 9.2 mg/L respectively. ECOSAR vinyl/allyl aldehyde estimates for the algal endpoint using the measured log Kow values as input give a 96h EC50 of 15.8 mg/L for the target substance and a value of 22.7 mg/L for the source substance. These estimates are considered reliable given that the experimental data for the source substance (Shisolia, 96-hr ErC50 of 21 mg/L) is in close agreement with the ECOSAR prediction. By comparing the ECOSAR estimates for the target and source substance, the log Kow difference results in a predicted algal 96h E50 value for Safranal that is 1.4 times lower than that for Shisolia. Applying this correction factor to the Shisolia measured values of 21mg/L (96h) and 18mg/L (72h), leads to corrected read-across estimates of 15 mg/L(96h) and 13mg/L (72h) for Safranal.
Conclusions:
In a guideline study, conducted according to GLP, the closely-related substance Shisolia was found to have an ErC50 (96 hr) of 21 mg/L and an ErC50 (72hr) of 18 mg/L.

The detailed information provided in the read-across justification document indicates that the aquatic ecotoxicity of Safranal (target substance) and Shisolia (source substance) are expected to be similar as a result of structural similarity, similar mechanistic profiles and similar physicochemical properties.

The measured log Kow of Safranal is slightly higher than that for Shisolia (2.7 versus 2.4). Thus, if aquatic toxicity tests were conducted on Safranal the determined response values might be slightly lower than those obtained for Shisolia (i.e. the strength of effects in the target substance might be slightly higher than observed for the source substance). By comparing the ECOSAR estimates for the target and source substance, the log Kow difference results in a predicted algal 96h E50 value for Safranal that is 1.4 times lower than that for Shisolia. Applying this correction factor to the Shisolia measured values of 21mg/L (96h) and 18mg/L (72h), leads to corrected read-across estimates of 15 mg/L(96h) and 13mg/L (72h) for Safranal. The latter value has been selected as the key result for the purpose of the chemical safety assessment.

In summary, read-across from Shisola is considered to give a reliable estimate of the algal EC50 for Safranal, which is a value of 15 mg/L (96h) and 13 mg/L (72h).

Description of key information

Based on a valid algal inhibition study on the registered substance (test item, Safranal), the 72h ErC10 and 72 ErC50 for 2,6,6-trimethylcyclohexa-1,3-dienecarbaldehyde was determined to be 3.88 and 15.5 mg/L, respectively. The latter is supported by data for a closely related analogue substance (test item, Shisola), which using the read-across approach gives an estimated 72h ErC50 of 13mg/L for 2,6,6-trimethylcyclohexa-1,3-dienecarbaldehyde. The determined values of ErC50 =15.5 mg/L and ErC10 = 3.88mg/L have been chosen as the key values for the purpose of the chemical safety assessment because they were obtained from the study on the registered substance itself.

Key value for chemical safety assessment

EC50 for freshwater algae:
15.5 mg/L
EC10 or NOEC for freshwater algae:
3.88 mg/L

Additional information

When the substance was first registered under REACH, the Lead Registrant used a read-across approach to address toxicity to aquatic algae and cyanobacteria. Since then a valid study on the registered substance has become available from a Joint Registrant. Both sets of information have been included in the dossier. The study performed on the registered substance is used as the key study and the read-across approach as supporting information.

 

KEY STUDY: The effect of the registered substance (test item Safanal, purity 95.4%) on the Growth of Freswater Alga (Pseudokirchneriella subcapitata) was followed over a period of 72 hours. The study was performed in compliance with following regulatory guideline: OECD Guidelines for the Testing of Chemicals, Section.2. Number 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” Adopted on 23 March 2006 Annexure 5 corrected: 28 July 2011. Based on the results of range finding test, a definitive test was conducted with the test concentrations of 1.0, 2.7, 7.0, 18.6 and 49.3 mg/L (geometric factor of 2.65) along with control and solvent control. For each test concentration three replicate were used and six replicates were maintained for control and solvent control group. At the end of 72 hour observation period, 0.78%, 10.56%, 18.39%, 44.21% and 94.53% growth inhibition and 3.43%, 35.77%, 53.79%, 84.98% and 99.57% yield inhibition was observed in the test concentrations of 1.0, 2.7, 7.0, 18.6 and 49.3 mg/L respectively. The effective concentrations based on growth rate and yield were calculated from the 72 hour algal cell count. The EC50/20/10 values based on the inhibition of growth rate and yield were calculated using probit analysis. A NOEC could not be determined.

The test item concentrations, control and solvent control were analyzed (0 and 72h samples) for definitive test. The test concentration was maintained within 80-120% of the nominal concentration throughout the exposure period. Therefore, the results were based on the nominal concentrations. The determined 72 hour ErC50 is 15.50 mg/L and the 72 hour EyC50 is 5.36 mg/L. The corresponding EC10 values are 3.88 and 1.33 mg/L.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate have been selected as the key values for classification purposes and risk assessment.

READ-ACROSS: The effect of the source substance on the growth of the green alga Pseudokirchneriella subcapitata was determined according to OECD guideline 201. It is a GLP compliant study conducted with a test material representative of the source substance and in compliance with agreed protocols, with no or minor deviations from the standard test. The study was considered valid.

As the test item is a volatile substance, the test was performed using flasks completely filled with test medium and tightly sealed with glass stoppers to avoid losses of test item. In order to evaluate the influence of the presence of light and algae on the stability of the test item, additional flasks containing the test medium were set up in a closed system. Three different incubation conditions, “Light with algae”, “Light without algae”, and “Darkness without algae”, were applied.

Over the test period of 96 hours the test item concentrations in the test media decreased. In the parallel stability study the main loss of test substance in the test water was attributed to adsorption to the growing algal biomass which accounted for approximately 13% decrease of the starting concentration over the period of 48 hours, 27% over 72 hours, and, 72% over 96 hours. This loss was considered in the calculation of the study endpoints, as the algal cells were, in effect, exposed to this complimentary fraction of test substance. Losses due to aqueous photolysis were only in the order of 5%. The study design covered the required exposure duration of 72/96 hours and included sufficient dose levels to enable the relevant determination of potency. The biological test results (based on mean measured test item concentrations) were as follows: the 72 hr ErC50 and ErC10 were determined to be 18 mg/L and 12 mg/L respectively; the 96 hr ErC50 and ErC10 were determined to be 21 mg/L and 13 mg/L respectively.

The measured log Kow of the target substance (Safranal, log Kow 2.7) is slightly higher than that for the source substance (Shisolia, log Kow 2.4). Thus, Safranal is expected to be slightly more toxic. This is supported by experimental fish LC50 values of 4.3 and 9.2 mg/L respectively (see the short-term toxicity to fish endpoint for details). ECOSAR vinyl/allyl aldehyde estimates for the algal endpoint using the measured log Kow values as input give a 96-hr EC50 value of 22.7 mg/L for the target substance and a value of 15.9 mg/L for the source substance. These estimates are considered reliable given that the experimental data for the source substance (21 mg/L) is in close agreement with the ECOSAR prediction (22.7 mg/L). By comparing the ECOSAR estimates for the target and source substance, the log Kow difference results in a predicted algal 96h E50 value for Safranal that is 1.4 times lower than that for Shisolia. Applying this correction factor to the Shisolia measured values of 21mg/L (96h) and 18mg/L (72h), leads to corrected read-across estimates of 15 mg/L(96h) and 13mg/L (72h) for Safranal.