Absolute of Boswellia Carterii (Burseraceae) obtained from exudate by hexane treatment and subsequent ethanol extraction

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-04-25 to 2017-11-16

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on January 10, 2017

Specific details on test material used for the study:

None.

Analytical monitoring:

yes

Details on sampling:

Duplicate samples for analysis were taken from the control and the limit test loading rate (from a replicate of each treatment without algae dedicated exclusively to chemical analyses) at the start and the end of the test. Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs. TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring. After having tried different methods for the preparation of the WAFs in the preliminary study, the protocol described hereafter has been selected for the final test.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 1 L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace). After heating of the test item sample to approx. 50°C for about 4 hours, the loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. Each glass flask was placed in a water bath for 10-15 minutes at approx. 50°C, followed by sonication for approx. 10 minutes. Based on experience on similar substances, the heating/sonication step is a method allowing to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 22 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were filtered and added into test flasks containing a fixed amount of inoculum (5.10^3 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, the solution was observed to be clear and colourless.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 -75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Stock culture: Algae stock cultures were started by inoculating growth medium (=test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2 °C.
- Pre-culture: 2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

23°C ± 2°C

pH:

Start : 8.23 (control) to 8.22 (test solution)
End : 9.75 (control) to 9.79 (test solution)
The pH level in the control varied more than 1.5 units at the end of the test (1.52 units of difference) this was not considered to have affected the integrity of the study.

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Limit test: 100 mg/L (loading rate)

Details on test conditions:

TEST DETAILS
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Initial cells density: An initial cell density of 5.10^3 cells.mL-1 using the exponentially growing pre-culture.
- Replicates: 6 replicates for the control and the loading rate at 100 mg.L-1. Moreover, replicates of each treatment without algae were prepared for chemical analyses.

GROWTH MEDIUM
- Standard medium used: yes: Original medium of OECD TG 201, Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: light intensity of 4,440-8,880 lux, did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED :
Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
Based on the results of a range-finding test, a limit test was performed at 100 mg.L-1 (loading).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

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: loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: loading rate

Details on results:

After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed, confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates. Based on these results, no ErLx and EyLx values could be determined due to the absence of effect at the tested loading rate.

Results with reference substance (positive control):

On August 21, 2017 (most recent test), the 72h-EC50 was 1.457 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Reported statistics and error estimates:

The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

Table pH-values during the final test

	Nominal concentration
	(mg test item.L^-1)*
	Control	100
Start t=0h	8.23	8.22
End t=72h	9.75	9.79

* WAF prepared at the given loading rate.

Table: Algal cell densities during the final test (expressed as density of algal cells/mL x 10⁴)

	Replicate	Nominal concentration (mg test item.L^-1)*
		Control	100
t=24h	1	4.4	5.6
	2	4.0	5.2
	3	7.2	6.4
	4	4.4	7.6
	5	4.8	4.4
	6	4.0	4.8
	Mean	4.8	5.7
	Std. Dev.	1.21	1.17
	% Inhib.	-	-7.6
t=48h	1	22.0	27.6
	2	21.2	20.4
	3	26.0	20.8
	4	18.0	29.2
	5	22.0	18.4
	6	20.0	21.6
	Mean	21.5	23.0
	Std. Dev.	2.66	4.34
	% Inhib.	-	-1.5
t=72h	1	86.0	93.6
	2	91.6	88.0
	3	84.4	92.4
	4	74.0	90.0
	5	93.6	81.2
	6	90.0	84.0
	Mean	86.6	88.2
	Std. Dev.	7.06	4.83
	% Inhib.	-	-0.4

* WAF prepared at the given loading rate.% Inhib.: %Inhibition of growth rate relative to the control determinedby the computer program ToxRat (8).At test start 5000algal cells.mL^-1were incubated; 6 replicates of the controls and 6 replicates of the limit test loading rate.Std. Dev.: standard deviation.

Validity criteria of the study

Cell density in controls: 173-fold increase within 72 hours. The specific growth rate was determined at 1.72 day^-1, so greater than 0.92 day^-1.

Coefficient of variation: 1.The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 26.82.The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.6%.

Thus the validity criteria were respected in this study.

Analytical results

Concentration of dissolved organic material in the controls and the WAFs was checked by TOC analysis at the start and at the end of the test. Even if the TOC analysis indicated that organic compounds were found at low value in the loading rates, analytical results showed that WAFs concentrations were overall stable between the start and the end of the exposure period.

It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture.Therefore, and since the test item was a UVCB substance, the results were based on thenominaltest loading rates.

Table: Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L^-1) - Final (limit) test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	0.88	0.47
Control	0.92	0.43
100	3.13	2.60
100	3.06	2.60

* WAF prepared at the given loading rate.

Physico-chemical parameter values throughout the test

Although the pH level in the control varied more than 1.5 units at the end of the test (1.52 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the addition of moresodium bicarbonate.

The mean light intensity was of 5320 lux (range: 4522-6118 lux), which remainedwithin the rangesprescribed by the study plan (range: 4440-8880 lux and not varying more than ± 15% from the average light intensity over the incubation area).

The temperature in the incubator was situated between 23.4 and 23.9°C throughout the test (average value: 23.8°C), and complied with the requirements as laid down in the study plan (23°C ± 2°C, constant within 2°C).

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg.L-1.

Executive summary:

A study was performed to assess the test item OLIBANUM RESINOIDE for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

A limit test was performed following the results of a range-finding test. Algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg test item/L and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the control and the limit test loading rate was checked by TOC analysisat the startand the end of the test.

Even if the TOC analysis indicated that organic compounds were found at low value in the loading rate of 100 mg/L, these analytical results showed that WAFs concentrations were overall stable between the start and the end of the exposure period.

After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed,confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates. Based on these results, no E_rL_x and E_yL_x values could be determined due to the absence of effect at the tested loading rate.

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg.L-1.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Read-across from a study performed on the analogue substance Resinoid of Boswellia Carterii (Burseraceae) obtained from exudate by hexane extraction.
The registered substance (target) and the source substance are natural extracts belonging to the group of Natural Complex Substances (NCS): UVCB sub-type 3, where the source is biological, and the process is refinement (ECHA Guidance on Identification and naming of substances under REACH, version 2.1 – May 2017, Section 4.3.1 and EFEO/IFRA Guidelines on substance identification and sameness of natural complex substances (NCS) under REACH and CLP, version of August 5, 2015).
The source substance Olibanum Resinoid and the registered substance have the same botanical origin: Boswellia Carterii (Burseraceae). They have the same related EC and CAS numbers as well as the same EC description. Compositions are not well defined but the registered substance is obtained from the source substance Olibanum resinoid by extraction with ethanol and elimination of the solvent. The source substance Olibanum resinoid contains all the constituents available from the extraction of the registered substance. Overall, the registered substance is expected to have its composition, and then its ecotoxicological behaviour, covered by Olibanum resinoid data. Therefore, we consider as reliable the read across from olibanum resinoid dossier to this dossier.

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

N/A

Vehicle:

no

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: loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: loading rate

Details on results:

After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed, confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates. Based on these results, no ErLx and EyLx values could be determined due to the absence of effect at the tested loading rate.

Results with reference substance (positive control):

On August 21, 2017 (most recent test), the 72h-EC50 was 1.457 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Reported statistics and error estimates:

The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

Table pH-values during the final test

	Nominal concentration
	(mg test item.L^-1)*
	Control	100
Start t=0h	8.23	8.22
End t=72h	9.75	9.79

* WAF prepared at the given loading rate.

Table: Algal cell densities during the final test (expressed as density of algal cells/mL x 10⁴)

	Replicate	Nominal concentration (mg test item.L^-1)*
		Control	100
t=24h	1	4.4	5.6
	2	4.0	5.2
	3	7.2	6.4
	4	4.4	7.6
	5	4.8	4.4
	6	4.0	4.8
	Mean	4.8	5.7
	Std. Dev.	1.21	1.17
	% Inhib.	-	-7.6
t=48h	1	22.0	27.6
	2	21.2	20.4
	3	26.0	20.8
	4	18.0	29.2
	5	22.0	18.4
	6	20.0	21.6
	Mean	21.5	23.0
	Std. Dev.	2.66	4.34
	% Inhib.	-	-1.5
t=72h	1	86.0	93.6
	2	91.6	88.0
	3	84.4	92.4
	4	74.0	90.0
	5	93.6	81.2
	6	90.0	84.0
	Mean	86.6	88.2
	Std. Dev.	7.06	4.83
	% Inhib.	-	-0.4

* WAF prepared at the given loading rate.% Inhib.: %Inhibition of growth rate relative to the control determinedby the computer program ToxRat (8).At test start 5000algal cells.mL^-1were incubated; 6 replicates of the controls and 6 replicates of the limit test loading rate.Std. Dev.: standard deviation.

Validity criteria of the study

Cell density in controls: 173-fold increase within 72 hours. The specific growth rate was determined at 1.72 day^-1, so greater than 0.92 day^-1.

Coefficient of variation: 1.The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 26.82.The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.6%.

Thus the validity criteria were respected in this study.

Analytical results

Concentration of dissolved organic material in the controls and the WAFs was checked by TOC analysis at the start and at the end of the test. Even if the TOC analysis indicated that organic compounds were found at low value in the loading rates, analytical results showed that WAFs concentrations were overall stable between the start and the end of the exposure period.

It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture.Therefore, and since the test item was a UVCB substance, the results were based on thenominaltest loading rates.

Table: Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L^-1) - Final (limit) test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	0.88	0.47
Control	0.92	0.43
100	3.13	2.60
100	3.06	2.60

* WAF prepared at the given loading rate.

Physico-chemical parameter values throughout the test

Although the pH level in the control varied more than 1.5 units at the end of the test (1.52 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the addition of moresodium bicarbonate.

The mean light intensity was of 5320 lux (range: 4522-6118 lux), which remainedwithin the rangesprescribed by the study plan (range: 4440-8880 lux and not varying more than ± 15% from the average light intensity over the incubation area).

The temperature in the incubator was situated between 23.4 and 23.9°C throughout the test (average value: 23.8°C), and complied with the requirements as laid down in the study plan (23°C ± 2°C, constant within 2°C).

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg.L-1.

Executive summary:

A study was performed to assess the test item OLIBANUM RESINOIDE for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

A limit test was performed following the results of a range-finding test. Algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg test item/L and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the control and the limit test loading rate was checked by TOC analysisat the startand the end of the test.

Even if the TOC analysis indicated that organic compounds were found at low value in the loading rate of 100 mg/L, these analytical results showed that WAFs concentrations were overall stable between the start and the end of the exposure period.

After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed,confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates. Based on these results, no E_rL_x and E_yL_x values could be determined due to the absence of effect at the tested loading rate.

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg.L-1.

The result of this study performed on an analogue is used for the registered substance.

Description of key information

Based on a read-across from an experimental algal growth inhibition study performed on the analogue substance "Resinoid of Boswellia Carterii (Burseraceae) obtained from exudate by hexane extraction" according to the OECD 201 guideline and under GLP conditions, the following results have been extrapolated to the registered substance:

-       72h-EL50 > 100 mg/L (based on growth rate and nominal loading rate)

-       72h-EL10 > 100 mg/L (based on growth rate and nominal loading rate)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

For that endpoint, a study on the registered substance was not available. Therefore, in order to assess the toxicity of the registered substance on freshwater algae, the results from an experimental OECD 201 study performed on the analogue substance "Resinoid of Boswellia Carterii (Burseraceae) obtained from exudate by hexane extraction" have been used.

In this study, the toxic effect of the test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a static limit test using Water Accommodated Fractions. The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 761/2009 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

A limit test was performed following the results of a range-finding test. Algal cells were exposed toWater Accommodated Fractions (WAFs)of the test item at a nominal loading rate of 100 mg test item/L and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the control and the limit test loading rate was checked by TOC analysisat the startand the end of the test.

Even if the TOC analysis indicated that organic compounds were found at low value in the loading rate of 100 mg/L, these analytical results showed thatWAFs concentrations were overall stablebetween the start and the end of the exposure period. After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed, confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates. Based on these results, no E_rL_xand E_yL_x values could be determined due to the absence of effect at the tested loading rate.

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg.L-1.

Besides one minor deviation (concerning some pH values) that did not affect the integrity of the study, the validity criteria were fulfilled and the study respected the requirements of the guideline. This study was therefore considered acceptable for that endpoint and the read-across justification is provided in the related iuclid study record.