2,3-dihydro-2,2,6-trimethylbenzaldehyde

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 09 ,2019 to February 21,2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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 19.01.2019 (0 hour) and 21.01.2019 (48 hour):
Control (T1, T2) samples were analyzed as such and T3, T5 and T7 samples were further diluted as below:

Sample ID Volume pipetted µL Volume diluted (mL) using acetonitrile
T3 (10 mg/L) 2500 5
T5 (31.3 mg/L) 800 5
T7 (98.2 mg/L) 500 10

Vehicle:

yes

Remarks:

solvent (acetone)

Details on test solutions:

Based on structural information provided by sponsor, it was determined that the test item was not soluble in ISO test water.
Hence the test item was dissolved in 0.1 ml/L to aid the test item to get soluble in ISO test water.

Test organisms (species):

Daphnia sp.

Details on test organisms:

Source: Bharathiyar University and sub cultured in house
Age: Aged less than 24 hours
Acclimatization: Acclimatization will be performed in the ISO Test water before test item exposure for 2 days (48 hours).
Photoperiod: 16-hour light and 8-hour dark cycle

Test type:

static

Water media type:

other: ISO Test water

Total exposure duration:

48 h

Hardness:

Measured once at the beginning of the acclimatization:
- Range finding test: 180 mg/L CaCO3
- Definitive test: 170 mg/L CaCO3

Test temperature:

Measured once at the beginning and end of the test:
- Range finding test: 20.1 to 20.4°C
- Definitive test: 20.1 to 20.3°C

pH:

Measured once at the beginning and end of the test:
- Range finding test: 7.7 to 8.0
- Definitive test: 7.7 to 8.0

Dissolved oxygen:

Measured once at the beginning and end of the test:
- Range finding test: 7.32 to 8.05 mg/L
- Definitive test: 7.78 – 8.35 mg/L

Conductivity:

Measured once at the beginning of the acclimatization:
- Range finding test: 312 µs cm-1
- Definitive test: 310 µs cm-1

Nominal and measured concentrations:

- Range Finding Test: At least five different test concentrations of 0.1, 1, 5, 10 and 100 mg/L with maximum the range of limit test concentration, 100 mg /L without a separating factor along with control and vehicle control was selected for range finding test.
- Definitive Test: Based on the results of the range finding test, a definitive test was performed with minimum of five concentration of 10, 17.7, 31.3, 55.5 and 98.2 mg/L with spacing factor in geometric series not exceeding 2.2. During the definitive test, the concentration verification of the test sample (low, mid and high) along with control and vehicle control at 0 hour and 48 hours were sent to Analytical chemistry for analysis.
- Justification for selection of concentrations: Concentrations selected for the test were based on the range finding test.

Details on test conditions:

Accommodation: 100 ml beaker
Medium: ISO Test water
Volume of medium: 50 mL

1- Range Finding Test
Control(s): 1- ISO Test water / 2- Solvent control
Treatment: Five, widely spaced Test concentrations (0.1, 1, 5, 10 and 100 mg/L)
Replication: 1 replicate was used for controls and treatments
Number of Daphnias per replicate: At least 5

2- Definitive Test
Control(s): 1- ISO Test water / 2- Solvent control
Treatment: At least Five Test concentrations (10, 17.7, 31.3, 55.5 and 98.2 mg/L)
Replication: 4
Number of Daphnias per replicate: 5
Feed: None
Aeration: None
Test Duration: 48 hours

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

22.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: with 95% confidence interval ranging from 18.50 mg/L to 26.96 mg/L.

Details on results:

- Range finding test
No cumulative immobilization was observed in daphnias treated with control, solvent control, 0.1, 1, 5 and 10 mg/L concentration whereas, 100% cumulative immobilization was observed in the tested concentrations of 100 mg/L for a period of 48 hours. No Daphnias exhibited any abnormal behaviour in control group(s) and in the tested concentrations for a period of 48 hours.
- Definitive test
No cumulative immobilization observed in control group(s) and 10 mg/L whereas, 40% and 70% in the tested concentrations of 17.7 and 31.3 mg/L for a period of 48 hours. The tested concentrations of 55.5 and 98.2 mg/L were observed 100% cumulative immobilization. No Daphnids exhibited any abnormal behaviour in control group(s) and in the tested concentrations of 10 mg/L for a period of 48 hours.
- Abnormal behaviour
No Daphnias exhibited any abnormal behaviour in control group(s) and in the tested concentrations for a period of 48 hours during range finding test and definitive test.
- Analytical
The test item concentrations and 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.

Results with reference substance (positive control):

EC50 value for 48 hours: 0.80 mg/L with 95% confidence interval ranging from 0.64 mg/L to 0.96 mg/L.

Reported statistics and error estimates:

The 48 hours EC50 value of Safranal was found to be 22.73 mg/L with 95% confidence interval ranging from 18.50 mg/L to 26.96 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the EC50 value for 48 hours of Safranal was found to be 22.73 mg/L with 95% confidence interval ranging from 18.50 mg/L to 26.96 mg/L.
The results observed in the present study meets all the validity criteria as per OECD 202 Test guideline.

Executive summary:

The objective of this study was to assess the Acute Immobilization Study of Safranal in Daphnia sp (Daphnia magna). The study was performed in compliance with following regulatory guideline: OECD Guideline for the Testing of Chemicals. Test Guideline 202: Daphnia sp. Acute Immobilization Test, Adopted: 13 April 2004.

A Range finding test was conducted with 5 groups at concentrations of 0.1, 1, 5, 10 and 100 mg/L along with a control and solvent control group(s) without test item. Each concentration contained one replicates and 5 Daphnias per replicate. No cumulative immobilization was observed in daphnias treated with control, solvent control, 0.1, 1, 5 and 10 mg/L concentration whereas, 100% cumulative immobilization was observed in the tested concentrations of 100 mg/L for a period of 48 hours. No Daphnias exhibited any abnormal behaviour in control group(s) and in the tested concentrations for a period of 48 hours.

Based on the results range finding test a definitive test was conducted with 6 groups at concentrations of 10, 17.7, 31.3, 55.5 and 98.2 mg/L along with a control group(s) without test item. Each concentration contained four replicates and 5 Daphnias per replicate. The treated Daphnias were maintained in a condition with temperature between 19.6°C and 20.3 °C and photoperiod was between 16 hours light and 8 hours dark for 48 hours. The pH range was 7.7 to 8.0 and Dissolved oxygen range was 7.78 to 8.35 mg/L. No cumulative immobilization observed in control group(s) and 10 mg/L whereas, 40% and 70% in the tested concentrations of 17.7 and 31.3 mg/L for a period of 48 hours. The tested concentrations of 55.5 and 98.2 mg/L were observed 100% cumulative immobilization. No Daphnias exhibited any abnormal behaviour in control group(s) and in the tested concentrations of 10 mg/L for a period of 48 hours.

Based on the results of this study, the EC50 value for 48 hours of Safranal was found to be 22.73 mg/L with 95% confidence interval ranging from 18.50 mg/L to 26.96 mg/L. The results observed in the present study meets all the validity criteria as per OECD 202 Test guideline.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From September 30, 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 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Daphnia magna

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Nominal and measured concentrations:

Nominal: 6.5, 13, 25, 50 and 100 mg/L.
Mean measured: --, 12, 23, 47 and 99 mg/L

The samples of the lowest nominal test concentration of 6.5 mg/L were not analyzed since the concentration was below the 48-hour NOEC determined in this test.

Details on test conditions:

As the test item is considered volatile, the test was performed using flasks tightly sealed and completely filled with test medium to avoid losses of test substance. Furthermore, since the photolytic stability of the test item was unknown, the test was performed in the dark to avoid potential photolytic degradation of the test item.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 27 - 37

Details on results:

In the analyzed test medium samples from the start and end of the first and second test medium renewal periods (duration 24 hours) the measured test item concentrations ranged between 83 and 99% of the nominal values. Consequently, the correct dosage of the test item could be verified and the test water renewal ensured sufficiently stable concentrations of the test item during the test.

Validity criteria fulfilled:

yes

Remarks:

The test is considered to be valid (no control daphnids showed immobilization or other signs of disease or stress and the dissolved oxygen concentration at the end of the test was ≥3 mg/L in all test media).

Conclusions:

In a guideline study, conducted according to GLP, Shisolia (GR-50-0091) was found to have an EC50 (48 hr) of 31 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 acute toxicity of the test item GR-50-0091 to Daphnia magna was determined in a 48-hour semi-static test according to the EU Commission Directive 92/69/EEC, Part C.2, the Commission Regulation (EC) No. 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004), as well as according to the OPPTS Guideline No. 850.1010 (Public Draft, April 1996).

The nominal test item concentrations tested were 6.5, 13, 25, 50 and 100 mg/L. Additionally, a control group was tested in parallel.

As the test item is a volatile substance, the test was performed using Erlenmeyer flasks completely filled with test medium that were tightly sealed with glass stoppers to avoid losses of test item (closed system). Furthermore, the test was performed in the dark to avoid photolytic degradation of the test item, in the event that the test substance may be susceptible to aqueous photolysis.

In the analyzed test medium samples from the start and end of the first and second test medium renewal periods (duration 24 hours) the measured test item concentrations ranged between 83 and 99% of the nominal values. Consequently, the correct dosage of the test item could be verified and the test water renewal ensured sufficiently stable concentrations of the test item during the test.

The biological results were related to the nominal and mean measured concentrations of the test item.

Nominal test concentration (mg/L)	Mean measured concentration of the test item (mg/L)	Mean measured concentration of the test item (% of nominal)
6.5	--	--
13	12	88
25	23	91
50	47	94
100	99	99

--: not analyzed

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the nominal test item concentration of 25 mg/L. At the two highest test concentrations of 50 and 100 mg/L, all test organisms were found to be immobile after 24 hours.

After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the nominal test item concentration of 13 mg/L. At the concentration of 25 mg/L, 2 daphnids were found to be immobile (10% immobilization). Although the same level of immobilization is tolerated by the guidelines also in the control, this immobilization was regarded as a toxic effect, as 3 other daphnids in this treatment showed symptoms of toxicity (reduced swimming ability) and at the next higher test concentration (50 mg/L) 100% of the daphnids were immobilized.

The biological test results (based on nominal and mean measured test item concentrations) were as follows:

	Based on Nominal	Based on Mean Measured
– 24-hour EC50: (95% confidence limits:	35 25 and 50	33 23 and 47	mg/L mg/L)
– 24-hour EC0:	25	23	mg/L
– 24-hour EC100:	50	47	mg/L
– 48-hour EC50: (95% confidence limits:	34 30 and 39	31 27 and 37	mg/L mg/L)
– 48-hour EC0 and  48-hour NOEC:	13	12	mg/L
– 48-hour EC100:	50	99	mg/L

The test is considered to be valid (no control daphnids showed immobilization or other signs of disease or stress and the dissolved oxygen concentration at the end of the test was ≥3 mg/L in all test media).                                                

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

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 target substance (Safranal) 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 daphnia endpoint, the subject of this read-across justification,
the ECOSAR vinyl/allyl aldehyde SAR estimates a 48-hr LC50 value of 37 mg/L for the source substance compared with a value of 23 mg/L for the target substance. These estimates are considered reliable given that the experimental data for the source substance (Shisolia, daphnia 48-hr EC50 of 31mg/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 for both substances 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.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: corrected for slight differences in log Kow

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

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). Thus, if aquatic toxicity tests were conducted on Safranal the determined response values might be slightly lower than those obtained for Shisolia. 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 daphnia endpoint using the measured log Kow values as input give a 48-hr LC50 value of 23 mg/L for the target substance and a value of 37 mg/L for the source substance. These estimates are considered reliable given that the experimental data for the source substance (Shisolia, daphnia 48-hr EC50 of 31mg/L) is in close agreement with the ECOSAR prediction. Based on ECOSAR estimates, the log Kow difference between the source and target substance results in a predicted daphnia LC50 value for Safranal that is 1.6 times lower than that predicted for Shisolia. Applying this correction factor of 1.6 to the measured value of 31mg/L for Shisolia, leads to a corrected read-across estimate of 19mg/L for Safranal. This value has been selected as the key result for the purpose of the CSA.

Conclusions:

In a guideline study, conducted according to GLP, the closely-related substance Shisolia was found to have an EC50 (48 hr) of 31 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, the strength of effects in the target substance might be slightly higher than observed for the source substance. Based on ECOSAR vinyl/allyl aldehyde estimates for the daphnia endpoint, the log Kow difference between the source and target substance results in a predicted LC 50 value for Safranal that is 1.6 times lower that predicted for the main constituents of Shisolia. Applying this correction factor to the measured value of Shisolia, leads to an estimated daphnia 48-hr LC50 of 19 mg/L for Safranal.

In summary, read-across from Shisola is considered to give a reliable estimate of the daphnia 48-hr EC50 for Safranal, which is a value of 19 mg/L.

Description of key information

Based on a valid short-term daphnia study on the registered substance (test item, Safranal ), the 48h EC50 for 2,6,6-trimethylcyclohexa-1,3-dienecarbaldehyde was determined to be 22.73 mg/L. This is supported by data for a closely related analogue substance (test item, Shisola), which using the read-across approach gives an estimated 48h EC50 for 2,6,6 -trimethylcyclohexa-1,3 -dienecarbaldehyde of 19 mg/L. The EC50 value of 22.73 mg/L has been chosen as the key value because it is obtained from the study on the registered substance itself.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

22.73 mg/L

Additional information

When the substance was first registered under REACH, the Lead Registrant used a read-across approach to address the short-term toxicity to aquatic invertebrates. 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 acute toxicity of the test item Safranal (purity 95.4%) to Daphnia magna was determined in a 48h test according to OECD Guideline for the Testing of Chemicals. Test Guideline 202: Daphnia sp. Acute Immobilization Test, Adopted: 13 April 2004. Based on the results of a range finding test, a definitive test was conducted with 6 groups at concentrations of 10, 17.7, 31.3, 55.5 and 98.2 mg/L along with a control group(s) without test item. Based on the results of stability analysis, a static test method was performed. The test item concentrations and control were analyzed for the definitive test. Since the test concentration was maintained within 80 -120% of the nominal concentration throughout the exposure period, the results were based on the nominal concentrations.

No cumulative immobilization was observed in control group(s) and 10 mg/L test concentration, whereas 40% and 70% immobilization was observed in the tested concentrations of 17.7 and 31.3 mg/L at 48 hours. At tested concentrations of 55.5 and 98.2 mg/L, 100% cumulative immobilization was observed. No Daphnids exhibited any abnormal behaviour in control group(s) and in the tested concentrations of 10 mg/L for a period of 48 hours. Based on the results of this study, the EC50 value for 48 hours of Safranal was found to be 22.73 mg/L with 95% confidence interval ranging from 18.50 mg/L to 26.96 mg/L. The results observed in the present study met all the validity criteria as per OECD 202 Test guideline.

READ-ACROSS: The analogue approach has been used which is based on the hypothesis that the source substance (Shisolia) and target substance (Safranal) are expected to 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). A detailed justification for the proposed read-across in-line with the ECHA RAAF guidelines is provided in the target endpoint record.

The acute toxicity of the source substance to daphnia was determined according to OECD guideline 202. 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 (no control daphnids showed immobilization or other signs of disease or stress and the dissolved oxygen concentration at the end of the test was ≥3 mg/L in all test media). A semi-static test with test medium renewal after 24 hours was performed to keep the concentrations of the test item in the test media as constant as possible during the test period of 48 hours. As the test item is volatile, the test was performed using flasks tightly sealed and completely filled with test medium to avoid losses of test substance. Furthermore, since the photolytic stability of the test item was unknown, the test was performed in the dark to avoid potential photolytic degradation of the test item. The study design covered the required exposure duration of 48 hours and included sufficient dose levels to enable the relevant determination of potency. The 48 hr EC50 was determined to be 31 mg/L (based on mean measured concentrations).

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 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 daphnia endpoint using the measured log Kow values as input give a 48-hr LC50 value of 23 mg/L for the target substance and a value of 37 mg/L for the source substance. These estimates are considered reliable given that the experimental data for the source substance (31mg/L) is in close agreement with the ECOSAR prediction (37mg/l). By comparing the ECOSAR LC50 estimates for the target and source substance, the log Kow difference results in a predicted daphnia LC50 value for Safranal that is 1.6 times lower than that for Shisolia. Applying this correction factor to the Shisolia measured value of 31mg/L, leads to a corrected read-across estimate of 19 mg/L for Safranal.