Reaction products from Nerolidol by dehydration

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - November 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Although a GLP compliant study conducted to OECD guidelines, all WAFs appeared turbid indicating that phase separation may have not been complete prior to drawing off the WAF and the presence of significant amounts of undissolved test item in the WAFs. The latter might explain the shallow does response observed. Hence the study is considered reliable with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Cas Number:

28973-99-1

Molecular formula:

C15H24

IUPAC Name:

(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Constituent 2

Reference substance name:

(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene

Cas Number:

25532-79-0

Molecular formula:

C15H24

IUPAC Name:

(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene

Constituent 3

Reference substance name:

(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)

Cas Number:

29837-07-8

Molecular formula:

C15H24

IUPAC Name:

(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)

Constituent 4

Reference substance name:

(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

EC Number:

242-582-0

EC Name:

(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Cas Number:

18794-84-8

Molecular formula:

C15H24

IUPAC Name:

7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Constituent 5

Reference substance name:

(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)

Cas Number:

53585-13-0

Molecular formula:

C15H24

IUPAC Name:

(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)

Constituent 6

Reference substance name:

(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)

Cas Number:

13062-00-5

Molecular formula:

C15H24

IUPAC Name:

(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)

Constituent 7

Reference substance name:

1-methyl-4-(6-methylhepta-1,5-dien-2-yl)

Cas Number:

869843-05-0

Molecular formula:

C15H24

IUPAC Name:

1-methyl-4-(6-methylhepta-1,5-dien-2-yl)

Constituent 8

Reference substance name:

(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Cas Number:

26560-14-5

Molecular formula:

C15H24

IUPAC Name:

(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Constituent 9

Reference substance name:

(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Cas Number:

28973-97-9

Molecular formula:

C15H24

IUPAC Name:

(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Constituent 10

Reference substance name:

1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene

Cas Number:

72345-84-7

Molecular formula:

C15H24

IUPAC Name:

1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene

Constituent 11

Reference substance name:

2,6,10-trimethyldodeca-2,6,9,11-tetraene

EC Number:

207-948-6

EC Name:

2,6,10-trimethyldodeca-2,6,9,11-tetraene

Cas Number:

502-61-4

Molecular formula:

C15H24

IUPAC Name:

3,7,11-trimethyldodeca-1,3,6,10-tetraene

Constituent 12

Reference substance name:

Likely sesquiterpene hydrocarbons

Cas Number:

n/a

Molecular formula:

C15H24

IUPAC Name:

Likely sesquiterpene hydrocarbons

Test material form:

liquid

Details on test material:

UVCB substance

Specific details on test material used for the study:

Batch number: VE00446520
Expiry date: 2017-12-27
Recommended storage: Dry, well-ventilated, preferably full, hermetically sealed, ambient temperature (10 – 30 °C or 50 – 85 °F), protected against light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Preparation of the water accomodated fraction:
Six water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 0.316 - 1.00 - 3.16 - 10.0 – 31.6 - 100 mg/L (spacing factor √10). The loading levels are based on the results of a preliminary range finding test (non GLP, closed bottles without headspace).
For the loading levels 1.00, 3.16, 10.0, 31.6 and 100 mg/L an appropriate amount of the test item was weighed. The test was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water. These dispersions were shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the aqueous phase or WAFs was removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fractions (WAF) were used in the test. The loading level 0.316 mg/L was prepared by diluting the WAF with a loading of 1.00 mg/L, since direct weighing of this loading was not technically feasible.

Test solutions

Details on test solutions:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble and multi-component test items, a modification of the standard method for the preparation of aqueous media will be performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Reason for the selection:
Pseudokirchneriella subcapitata is a suitable green alga species of the test organism according to the guideline.
Origin:
Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches Institut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen
Cultivation at test facility:
Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35-70 μE·m-2·s-1 for 24 hours per day.
Culture medium:
Nutrient medium Z according to LÜTTGE et al. (1994)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

min.: 22.0°C max.: 22.5°C

pH:

min.: 7.82 max.: 9.30

Nominal and measured concentrations:

Six water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 0.316 - 1.00 - 3.16 - 10.0 - 31.6 – 100 mg/L (spacing factor √10). The loading levels are based on the results of a preliminary range finding test (non GLP, closed bottles without headspace).

Details on test conditions:

Due to the volatility of the test item, closed glass flasks without headspace were used to reduce contact with air and losses of test item by evaporation.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on WAF loading levels

Remarks:

33% inhibition at highest loading level of 100mg/L

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 v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. 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 presented in the above "effects concentration" table.

The mean % inhibition based on growth rate at the six loading levels of 100, 31.6, 10.0, 3.16, 1.00 and 0.316 mg/L were respectively 33%, 19%, 18%, 11%, 15% and 10%. Thus the ErL50 is > 100 mg/L and the NOEC < 0.316 mg/L. The ErL510 was also determined to be < 0.316 mg/L.

All WAFs appeared turbid indicating that phase separation may have not been complete prior to drawing off the WAF and the presence of significant amounts of undissolved test item in the WAFs. The latter might explain the shallow does response observed. Hence the study is considered reliable with restrictions and the chronic endpoints (NOEL and EL10) are not considered adequate for the purposes of classification.

The measured concentrations of the test item at the start of the exposure increased with the applied dose. The initial measured concentrations for the six WAFs were 0.259, 0.674, 2.53, 10.3, 12.4 and 15.3 mg/L. At test end, the test item concentrations were in the range of 44 to 77% of initial. Given that all efforts were made to minimise loss (sealed system) and that WAFs were used because of the low aqueous solubility and complex nature of the test item, it is considered appropriate to express the effects based on loading levels.

Results with reference substance (positive control):

The health of the algae culture and the overall quality of the test organism is monitored regularly by using the reference toxicant, potassium dichromate. Reference tests performed from 2017-04-18 to 2017-04-21 (with headspace) and 2016-10-10 to 2016-10-13 (without headspace) resulted in an ErC50 (0-72h) of 0.559 mg/l and 0.811 mg/L respectively. The results were within the normal range for this reference item; 0.773 ± 0.540 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, BISABOLENE was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal item loadings): The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 100 mg/L and 4.13 (0.630 – 17.3) mg/L, respectively. The EL10-values with 95% confidence intervals for both inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were < 0.316 mg/L. The NOEL-values for both inhibition of growth rate and yield after 72 hours were < 0.316 mg/L, respectively.

Executive summary:

In this study under OECD guideline No. 201, BISABOLENEwas found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal item loadings):

The EL₅₀-values with 95% confidence intervals for inhibition of growth rate (E_rL₅₀) and yield (E_yL₅₀) after 72 hours were > 100 mg/L and 4.13 (0.630 – 17.3) mg/L, respectively.

The EL₁₀-values with 95% confidence intervals for both inhibition of growth rate (E_rL₁₀) and yield (E_yL₁₀) after 72 hours were < 0.316 mg/L.

The NOEL-values for both inhibition of growth rate and yield after 72 hours were < 0.316 mg/L, respectively.

The study was conducted in a closed system (sealed glass flasks) without headspace, due to the volatility of the test item, to reduce contact with air and losses of the test item by evaporation. Due to the low aqueous solubility and complex nature of the test item for the purposes of the test, the test item was prepared as water accommodated fractions (WAFs).

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 v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50 (equivalent to ErL50 for a WAF study). Thus the ErL50 of > 100mg/L has been used for classification purposes.

The chronic endpoints (NOEL and EL10) are not considered adequate for the purposes of classification due to the shallow dose response curve, which might be attributed to the turbid nature of the WAFS, and because values are less than the lowest loading level.