Rosemary, ext.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

key study

Study period:

2014-07-21 to 2014-07-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Calculation method is used ; calculation method applicable for the endpoint.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

Calculation method

Principles of method if other than guideline:

The acute toxicity to fish was determined using a validated QSAR for the Mode of Action in question. The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.
Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.
The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF.
The method has been validated using data derived from 96-hour LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the lethal loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction.

GLP compliance:

no

Analytical monitoring:

not required

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

LL50 (lethal loading rate of WAF)

Post exposure observation period:

not applicable

Hardness:

Hardness is not a necessary component of the WAF calculation

Test temperature:

The Temperature is not a necessary component of the WAF calculation but extremely low or high temperatures could influence the solubility of certain constituents. Therefore, the calculation method is considered acceptable to determine LL50s for fish between 12 and 28°C.

pH:

The pH is not a necessary component of the WAF calculation

Dissolved oxygen:

The oxygen concentration is not a necessary component of the WAF calculation

Salinity:

Salinity is not a necessary component of the WAF calculation. However as the fish QSAR for the constituents calculation was based on data from freshwater studies, the resulting calculation is considered valid for freshwater fish

Nominal and measured concentrations:

The calculation determines measured concentrations

Details on test conditions:

calculation method

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

3.9 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: typical composition (please refer to confidential section)

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

3.4 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: theoretical worst case composition (please refer to confidential section)

Details on results:

not applicable

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Sublethal observations / clinical signs:

Table 1: Expected concentrations at this 96h-LL50 (composition 1: typical composition)

constituents	concentration in the WAF (mg.L-1)
cineol-1,8	0.68
limonene	0.15
α-pinene	0.92
camphor	0.67
terpineol	0.11
β-pinene	0.10
p-cymene	0.07
camphene	0.38

Table 2: Expected concentrations at this 96h-LL50 (composition 2: theoretical worst case composition)

constituents	concentration in the WAF (mg.L-1)
cineol-1,8	0.82
limonene	0.24
α-pinene	0.85
camphor	0.65
terpineol	0.18
β-pinene	0.14
p-cymene	0.12
camphene	0.37

Validity criteria fulfilled:

yes

Conclusions:

96h-LL50 for typical composition of Rosemary oil = 3.9 mg test item/L and 96h-LL50 for theoretical worst case composition of Rosemary oil = 3.4 mg test item/L

Executive summary:

Rosemary oil is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage and its reported variation of each constituent is known.

Its acute toxicity to fish property has been investigated using an in-house calculation method that replaces an OECD 203 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions). Two theoretical compositions have been investigated, the “typical” composition proposed by the Lead registrant and a theoretical worst case composition that maximizes the concentration of the most toxic constituents.The acute toxicity to fish was determined using a calculation method for Mode of Action 1 (non-polar narcotics) for each individual constituent present in the mixture. This algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004).

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.

In the calculation the second step is to remove this non-bioavailable fraction. Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. These two reasons explain why ecotoxicity values from WAF studies are always higher for non-polar narcotic mixtures than the calculated values from CLP additivity calculation

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or predicted using the iSafeRat QSAR model. Each value has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF. Using this approach, the 96-h LL50 for fish was 3.9 mg test material/L for the typical composition of Rosemary oil, and the 96-h LL50 was 3.4 mg test material/L for the theoretical worst case composition. These LL50 were based on mortality. 

 

Based on the results of this study, Rosemary oil would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

This toxicity study is acceptable and can be used for that endpoint.

 

Results Synopsis

Test Type: Calculation method

LL50: 3.9 mg test material/L for typical composition

LL50: 3.4 mg test material/L for theoretical worst case composition

 

Description of key information

Rosemary oil is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage and its reported variation of each constituent is known. The acute toxicity to fish property has been investigated using an in-house calculation method that mimics an OECD 203 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions). The acute toxicity to fish was determined using a calculation method for Mode of Action 1 (non-polar narcotics) for each individual constituent present in the mixture. This algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004). Further to this, the lethal loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics first to determine the analysable fraction (the concentration which should be analysable in a WAF study) and then excluding the non-bioavailable fraction of the remaining constituents. The remaining, bioavailable fraction corresponds to the lethal loading value of the mixture. This approach has been validated using data derived from 96-hour LL50 tests on fish with similar complex substances (OECD 203 study and the OECD guidance document on toxicity testing for difficult substances and mixtures No. 23, i.e. WAF conditions). Two theoretical compositions have been investigated, the “typical” composition proposed by the Lead registrant and a theoretical worst case composition that maximizes the concentration of the most toxic constituents. The 96-h LL50 was 3.9 mg test material/L for the typical composition of Rosemary oil, and the 96-h LL50 was 3.4 mg test material/L for the theoretical worst case composition. LL50s were based on mortality. Conclusion: The 96-h LL50 for fish was calculated at 3.9 mg test material/L for the typical composition and at 3.4 mg test material/L for theoretical worst case composition.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

3.4 mg/L

Additional information

Rosemary oil is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage of each constituent is known. Therefore, it has been decided that the ecotoxicity of Rosemary oil will be derived from knowledge of the constituents, constituent approach

The mixture ecotoxicity properties may be derived from the ecotoxicity of the individual constituents (table 1) using the CLP additivity calculation approach. However, CLP additivity approach is calculated on the basis that all the substances are at their maximum solubility and it has been observed that CLP additivity calculations for mode of action 1 compounds are unreasonably conservative when compared to classic WAF studies. This has been proved in a number of cases for natural complex substances. Indeed, natural extract compositions are a mixture of hydrophilic alcohol molecules and hydrophobic terpene molecules. Therefore, when a WAF is performed most of substances fully dissolved in the aqueous phase are the hydrophilic fraction while the hydrophobic fraction (the more toxic elements for MOA 1 substances) may be below their water solubility value.

 

The acute toxicity to fish property for Rosemary oil has been investigated using a calculation method (iSafeRat WAF module for mixture Toxicity calculation) that mimics an OECD 203 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions). Two theoretical compositions have been investigated, the “typical” composition proposed by the Lead registrant and a theoretical worst case composition that maximizes the concentration of the most toxic constituents. The acute toxicity to fish was determined using a calculation method for Mode of Action 1 (non-polar narcotics) for each individual constituent present in the mixture. This algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004).

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.

In the calculation the second step is to remove this non-bioavailable fraction. Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. These two reasons explain why ecotoxicity values from WAF studies are always higher for non-polar narcotic mixtures than the calculated values from CLP additivity calculation.

 

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or predicted using the iSafeRat QSAR model. Each value has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF. Using this approach, the 96-h LL50 for fish was 3.9 mg test material/L for the typical composition of Rosemary oil, and the 96-h LL50 was 3.4 mg test material/L for the theoretical worst case composition. These LL50 were based on mortality. 

 

The in-house calculation has been designed to mimic the behavior of the mixture in a WAF test where the final toxicity is calculated by additivity of the residual bioavailable fraction of each constituent. This approach has been validated using a Natural Complex Substance similar to Rosemary oil as presented in the position paper attached to the dossier.

 

Based on the results of this study, Rosemary oil would not be classified as acutely toxic to aquatic organisms in accordance with the classification of the CLP.

This toxicity prediction has been validated and is considered acceptable to fulfill the fish toxicity endpoint.

 

Table 1 Rosemary oil, constituents data used to derive Rosemary oil Ecotoxicity value.

 

constituent	typical composition (%)	worst case concentration (%)	96-fish LC50 (mg/L)	96-fish LC50 reference
α-pinene	23.57	25.00	0.280 [0.259 – 0.303]	literature source
cineol 1,8	17.54	24.00	57 [32 – 100]	literature source
limonene	3.83	7.00	0.71 [618 – 839]	literature source
terpineol	2.70	5.40	60.93 [49.60 – 74.89]	iSafeRat® prediction
para-cymene	1.87	3.50	1.63 [1.21 – 2.20]	iSafeRat® prediction
β-pinene	2.63	4.00	0.44 [0.29 – 0.66]	iSafeRat® prediction
Camphene	9.70	11.00	0.44 [0.29 – 0.66]	iSafeRat® prediction
camphor	17.29	19.00	22.01 [18.36 – 26.38]	iSafeRat® prediction