Caryophyllene, Reaction product with Acetic anhydride and acetic acid

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 04-October 10, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[1R-(1α,2α,5β,8β)]-4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-yl acetate

EC Number:

260-555-1

EC Name:

[1R-(1α,2α,5β,8β)]-4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-yl acetate

Cas Number:

57082-24-3

Molecular formula:

C17H28O2

IUPAC Name:

4,4,8-trimethyltricyclo[6.3.1.0~2,5~]dodec-1-yl acetate

Constituent 2

Reference substance name:

1-((1S,2S,5R)-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-8-en-9-yl)ethan-1-one

Molecular formula:

C17H26O

IUPAC Name:

1-((1S,2S,5R)-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-8-en-9-yl)ethan-1-one

Constituent 3

Reference substance name:

(3S,3aS,7R,9aS)-1,1,7-trimethyldecahydro-3a,7-methanocyclopenta[8]annulen-3-yl acetate

Cas Number:

77346-43-1

Molecular formula:

C17H28O2

IUPAC Name:

(3S,3aS,7R,9aS)-1,1,7-trimethyldecahydro-3a,7-methanocyclopenta[8]annulen-3-yl acetate

Constituent 4

Reference substance name:

1-((1S,2S,5R,9S)-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-7-en-9-yl)ethan-1-one

Cas Number:

Not applicable

Molecular formula:

C17H26O

IUPAC Name:

1-((1S,2S,5R,9S)-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-7-en-9-yl)ethan-1-one

Constituent 5

Reference substance name:

4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-ol

EC Number:

207-458-2

EC Name:

4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-ol

Cas Number:

472-97-9

Molecular formula:

C15H26O

IUPAC Name:

4,4,8-trimethyltricyclo[6.3.1.0~2,5~]dodecan-1-ol

Constituent 6

Reference substance name:

(3S,3aS,7R,9aS)-1,1,7-trimethyldecahydro-3a,7-methanocyclopenta[8]annulen-3-ol

Cas Number:

62560-57-0

Molecular formula:

C15H26O

IUPAC Name:

(3S,3aS,7R,9aS)-1,1,7-trimethyldecahydro-3a,7-methanocyclopenta[8]annulen-3-ol

Constituent 7

Reference substance name:

2,2,4,7a-tetramethyl-1,2,3,6,7,7a-hexahydro-1,3a-ethanoindene

Cas Number:

4545-68-0

Molecular formula:

C15H24

IUPAC Name:

2,2,4,7a-tetramethyl-1,2,3,6,7,7a-hexahydro-1,3a-ethanoindene

Constituent 8

Reference substance name:

not applicable

Cas Number:

not applicable

Molecular formula:

not applicable

IUPAC Name:

not applicable

Test material form:

liquid

Specific details on test material used for the study:

Name (as stated in the report): VETYNAL
Batch: VE00473596
Expiration date: March 05, 2019
Purity / Composition Type: UVCB (about 90 %, sum of main constituents)

Sampling and analysis

Analytical monitoring:

yes

Remarks:

GC - FID

Details on sampling:

Analysis of the Test Item Concentrations

The test item gave a chromatographic profile consisting of several peaks, thereof two main
peaks which represent about 75% of the test item (area %). The analytical characterization of
the WAFs was based on these two peaks.
For this, duplicate samples were taken from each test loading rate and the control at the start
and at the end of the test. For sampling from the aged test media, the contents of the respective
replicates were combined prior to sampling.
All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on analytical
pre-experiments for investigation of the storage stability, the analyzed components of the test
item were found to be stable in the test water under these storage conditions.

The concentrations of the two main constituents of VETYNAL were analyzed in one of the
duplicate test medium samples taken from all treatments and both sampling dates (0 and
48 hours).

Test solutions

Vehicle:

no

Details on test solutions:

For the preparation of the test media, the test item was carefully applied (pipetted) onto the surface of test water. The test item volumes were calculated, considering the density of the test item of 0.9896 g/cm3 (at 20 °C).

A stirring period of 48 hours at room temperature in the dark was applied. According to the stirring experiment, 48 hours was considered to be an appropriate stirring period to reach a sufficient concentration of dissolved test item in the test media. The equilibrated aqueous phases with the respective loading rates, containing dissolved test item only (clear solutions), were tested as WAFs. Due to technical reasons (operational
constraints on the precision of direct weighing of test item), the WAF with the lowest loading rate of 1.0 mg/L was prepared as a dilution of the equilibrated aqueous phase of the WAF with the loading rate of 3.2 mg/L. Additionally, a control (test water only) was run in parallel.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (originally from the Daphnia Collection of the University of Basel/Switzerland in 2015) is successfully bred in IES Ltd Laboratories. The cultivation of the parental daphnids
is performed in reconstituted water of the quality identical to the water quality used in the tests (with respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.

During breeding, daphnids were generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany) and
cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-GmbH, 49324 Melle/Germany).
At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in March 2017 (24-hour EC50: 1.2 mg/L, IES Laboratories Study 20170076) showed that
the sensitivity of the test organisms was within the range given by the guideline (24-hour EC50: 0.60-2.1 mg/L).

Study design

Test type:

static

Water media type:

other: Reconstituted test water (ISO Test water)

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

2.5 [mmol/L];
250 as CaCO3 [mg/L]

Test temperature:

21 °C

pH:

between 7.7 and 7.8

Dissolved oxygen:

> = 8.1 mg/L

Nominal and measured concentrations:

The test item gave a chromatographic profile consisting of several peaks, thereof two main
peaks which represent about 75% of the test item (area %). The analytical characterization of
the WAFs was based on the sum of these two peaks.

At the start of the test, the measured test item concentrations (based on the measurement of the
two main peaks) in the test media with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were
0.35, 1.1, 1.1, 1.4 and 1.4 mg/L, respectively. At the four highest loading rates, the analyzed
test item concentrations were in the same range of 1.1 to 1.4 mg/L, showing a plateau and
demonstrating that the maximum concentration (sum of peaks) of dissolved test item was
reached under the conditions of the test. At the lowest WAF, the analyzed test item
concentration reflected the gradation of the WAF with the spacing factor of 3.2. The test item
concentrations remained stable over 48 hours. 84 to 104 % of the initially measured values were
determined at the end of the test.

Since VETYNAL consists of several constituents with different solubilities and the analytical
method is based only on the two main constituents, the absolute amount of test item could not
be analytically determined and the measured test item concentrations should be regarded as
approximated values. Therefore, all reported biological results were based on the loading rates
of the test item. This procedure of evaluation of data of WAFs is based on recommendations of
the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and
Mixtures, 2000.

Details on test conditions:

The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 21 °C.

The daphnids were not fed during the test. A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was between 15 and 17 μmol m-2 s-1.

Reference substance (positive control):

yes

Remarks:

Potassium DiChromate tested as a toxicity reference standard twice a Year

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

6.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 4.4-8.6 [mg/L]

Remarks:

Based on Loading Rate

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Based on Loading Rate

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Based on Loading Rate

Details on results:

The analytical characterization of the WAFs was based on the sum of peaks of the two main constituents. At the start of the test, the measured test item concentrations in the test media with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were 0.35, 1.1, 1.1, 1.4 and 1.4 mg/L, respectively (see Table 7 in Appendix 1). At the four highest loading rates, the analyzed test item concentrations were in the same range of 1.1 to 1.4 mg/L, showing a plateau and demonstrating that the maximum concentration (sum of peaks) of dissolved test item was reached under the conditions of the test. At the lowest WAF, the analyzed test item concentration reflected the gradation of the WAF with the spacing factor of 3.2.

The test item concentrations (based on the measurement of the two main peaks) remained stable over 48 hours. 84 to 104 % of the initially measured values were determined at the end of the test.

Since VETYNAL consists of several constituents with different solubilities and the analytical method is based only on the two main constituents, the absolute amount of test item could not be analytically determined and the measured test item concentrations should be regarded as approximated values. Therefore, all reported biological results were based on the loading rates
of the test item. This procedure of evaluation of data of WAFs is based on recommendations of the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

A clear dose-response effect was observed during the study. During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the loading rate of 3.2 mg/L. At the three highest loading rates of 10, 32 and 100 mg/L, 15, 30 and 40 % of the daphnids were found to be immobile, respectively. Reduced swimming activity was also observed in some of the remaining mobile daphnids at the loading rates of 32 and 100 mg/L.

The 24-hour EL50 of the test item was determined to be greater than 100 mg/L. The 24-hour EL0 and NOELR were at the loading rate of 3.2 mg/L. The 24-hour EL100 was greater than 100 mg/L.

After 48 hours exposure, no immobilized test organisms were determined in the control and at the loading rate of 1.0 mg/L. At the next three loading rates of 3.2, 10 and 32 mg/L, 20, 80 and 95 % of the daphnids were found to be immobile, respectively. Reduced swimming activity was also observed in some of the remaining mobile daphnids at these loading rates. At the highest loading rate of 100 mg/L, 100 % of the daphnids were found to be immobile

The 48-hour EL50 of the test item was calculated to be 6.1 mg/L with 95% confidence limits of 4.4 and 8.6 mg/L. The 48-hour EL0 and NOELR were at the loading rate of 1.0 mg/L. The 48-hour EL100 was 100 mg/L.

No remarkable observations were made concerning the appearance of the test media. All test media remained clear solutions throughout the test. At the beginning and end of the test, the pH values of the test media were between 7.7 and 7.8 (See on Table 2 of report). The dissolved oxygen concentrations in the test media and control were at least 8.1 mg/L and the water temperature was maintained at 21 °C during the test.

Results with reference substance (positive control):

The result of the latest positive control
test in March 2017 (24-hour EC50: 1.2 mg/L, IES Laboratories Study 20170076) showed that
the sensitivity of the test organisms was within the range given by the guideline (24-hour EC50:
0.60-2.1 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress.

Conclusions:

The 48-hour EL50 of the test item was calculated to be 6.1 mg/L with 95% confidence limits of
4.4 and 8.6 mg/L. The 48-hour EL0 and NOELR were at the loading rate of 1.0 mg/L.
The 48-hour EL100 was 100 mg/L.

Executive summary:

The acute toxicity of the test item VETYNAL to Daphnia magna was determined in a 48-hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.

VETYNAL consists of several constituents with different solubilities and is characterized as a UVCB (a chemical substance of Unknown or Variable Composition, complex reaction products and Biological materials). Therefore, Water Accommodated Fractions (WAFs) with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were tested to assess the toxicity of the test item to daphnids.

As the test item is a volatile substance, the test was performed using glass tubes completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system).

Since the test item is a liquid with low water solubility, the slow-stirring method (to avoid formation of micro emulsions) was applied for preparation of the individual saturated test item solutions. For this, the test item with the density of 0.9896 g/cm3 (at 20 °C) was carefully

applied (pipetted) on to the surface of the test water at the loading rates of 3.2, 10, 32 and 100 mg/L. Thereafter, slow-stirring was applied for 48 hours in closed vessels to reach a maximum concentration of dissolved test item in the test water. The stirring vessels were nearly completely filled (a small headspace had to be included as the test item was floating on the water surface) and tightly sealed with glass stoppers to avoid losses of the volatile test item during stirring.

After this treatment the lower parts of the equilibrated test media were carefully harvested from the stirring vessel through a tap at the bottom of the vessels. The WAF with the lower loading rate of 1.0 mg/L was prepared as a dilution of the WAF with the loading rate of 3.2 mg/L since it was technically infeasible to directly weight the required amount of test item. Additionally, a control (test water without test item) was tested in parallel.

The preparation of the test media was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

The test item gave a chromatographic profile consisting of several peaks, thereof two main peaks which represent about 75% of the test item (area %). The analytical characterization of the WAFs was based on the sum of these two peaks.

At the start of the test, the measured test item concentrations (based on the measurement of the two main peaks) in the test media with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were 0.35, 1.1, 1.1, 1.4 and 1.4 mg/L, respectively. At the four highest loading rates, the analyzed test item concentrations were in the same range of 1.1 to 1.4 mg/L, showing a plateau and demonstrating that the maximum concentration (sum of peaks) of dissolved test item was reached under the conditions of the test. At the lowest WAF, the analyzed test item concentration reflected the gradation of the WAF with the spacing factor of 3.2. The test item concentrations remained stable over 48 hours. 84 to 104 % of the initially measured values were determined at the end of the test.

Since VETYNAL consists of several constituents with different solubilities and the analytical method is based only on the two main constituents, the absolute amount of test item could not be analytically determined and the measured test item concentrations should be regarded as approximated values. Therefore, all reported biological results were based on the loading rates of the test item. This procedure of evaluation of data of WAFs is based on recommendations of the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

A clear dose-response effect was observed during the exposure period: At the three highest WAFs with the loading rates of 10, 32 and 100 mg/L, an immobility of 80, 95 and 100 % was observed for the daphnids after 48 hours exposure, respectively. This result is conform with the analytical results which showed the maximum concentration of dissolved test item at the start of the renewal periods (range 1.1 to 1.4 mg/L) at these WAFs. At the lower WAFs of 1.0 and 3.2 mg/L, 0 and 20 % immobilization was observed, respectively.

ELx Values Immobility: Based on Loading Rates of the Test Item [mg/L]:

24-hour EL50 >100

24-hour EL0 and NOELR 3.2

24-hour EL100 >100

48-hour EL50 6.1

(95% confidence limit (4.4-8.6)

48-hour EL0 and NOELR 1.0

48-hour EL100 100

Validity criteria:

The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discolouration or unusual behaviour such as trapping at the surface of water). Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in all test vessels.