Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

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:

(Q)SAR

Adequacy of study:

key study

Study period:

07 - 10 November 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Guideline:

other: REACH guidance on QSARs R.6, OECD Guideline 202 (Acute Daphnia sp. Immobilisation Test), with modifications from OECD Series on Testing and Assessment no. 23 on ecotoxicity testing of difficult substances.

Principles of method if other than guideline:

The QSAR method was validated using data from tests preformed using the OECD document 23 recommnedations for poorly water soluble complex mixtures. The QSAR is thus validated ot be compliant inline with such methods/recommendations.

Specific details on test material used for the study:

The QSAR method was validated using data from tests preformed using the OECD document 23 recommnedations for poorly water soluble complex mixtures. The QSAR is thus validated not be compliant inline with such methods/recommendations.

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

6.8 mg/L

Remarks on result:

other: QSAR predicted value

Details on results:

This approach uses a thermodynamically based module to calculate the water concentrations and the individual toxicity of each of the constituent is first derived from a QSAR model validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004). Then the effective loading rate of the mixture to the test organisms (i.e. Daphnia magna) is determined as a Water Accommodated Fraction (WAF) test (OECD, 2000).

Conclusions:

The 48 hour EL50 to Daphnia sp. of the reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene, estimated from the iSafeRat v.2.2 QSAR inputs from each constituent, calculations based on thermodynamics of each constituent and there eventual contribution to the toxicity was 6.8 mg/L.

The endpoint value is considered valid for use in risk assessment (including PBT assessment) and classification and labelling. The prediction is adequate as contributing data for the Classification and Labelling or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006: Annex XI Section 1.3.

Executive summary:

This approach uses a thermodynamically based module to calculate the water concentrations and the individual toxicity of each of the constituent is first derived from a QSAR model validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004). Then the effective loading rate of the mixture to the test organisms (i.e. Daphnia magna) is determined as a Water Accommodated Fraction (WAF) test (OECD, 2000).

The inputs into the model all came from reliable QSAR estimations. The endpoint value is considered valid for use in risk assessment (including PBT assessment) and classification and labelling. The prediction is adequate as contributing data for the Classification and Labelling or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006: Annex XI Section 1.3.

The 48 hour EL₅₀ for the test substance was estimated to be 6.8 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 July - 02 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines (OECD 202) and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Protected from extremes of temperature, under nitrogen.
- Stability under test conditions: 52 - 74 % of applied limonene (used as an indicator of test items stability) recovered at 48 h.
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion. 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 5 L. A magnetic stirring bar was placed in each mixing vessel and 5.2 to 5.5 L of test water (depending on the brim capacity of the bottles) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. 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 24 ± 2 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 directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless at all loading rates.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: as above
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): as above

OTHER SPECIFICS: n/a

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2.0, 3.0, 4.5, 6.8 and 10 mg/L.
- Sampling method: Single samples for analysis were taken from the control and all test concentrations at the start of the test (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). Chemical analyses of constituent 1 ((4R)-4-isopropenyl-1-methylcyclohexene; limonene), one of the two major was performed.
- Sample storage conditions before analysis: not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion. 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 5 L. A magnetic stirring bar was placed in each mixing vessel and 5.2 to 5.5 L of test water (depending on the brim capacity of the bottles) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. 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 24 ± 2 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 directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless at all loading rates.
- Eluate: Elendt M4 medium
- Controls: Elendt M4 medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, clone 5
- Justification for species other than prescribed by test guideline: n/a
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Age of parental stock (mean and range, SD): < 24 h
- Feeding during test: No
- Food type: n/a
- Amount: n/a
- Frequency: n/a

ACCLIMATION
- Acclimation period: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Pseudokirchneriella subcapitata - up to 0.1 - 0.2 mg C/Daphnia/day.
- Feeding frequency: 3 times/week
- Health during acclimation (any mortality observed): Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.

QUARANTINE (wild caught)
- Duration: n/a
- Health/mortality: n/a

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:

The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

ca. 250 mg/L (as CaCO3)

Test temperature:

20.6 - 20.9 ºC

pH:

8.08 - 8.16

Dissolved oxygen:

8.33 - 8.69

Salinity:

n/a

Conductivity:

not reported

Nominal and measured concentrations:

Nominal: 2.0, 3.0, 4.5, 6.8 and 10 mg/L.
Mean measured: 74, 71, 62, 63 and 52 % of nominal (based on limonene concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: no headspace
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): re-newed daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): n/a
- Biomass loading rate: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Conductivity: not reported
- Salinity: not reported
- Culture medium different from test medium: n/a
- Intervals of water quality measurement: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all test concentrations and control.

OTHER TEST CONDITIONS
- Adjustment of pH: n/a
- Photoperiod: 16 h: 8 h (light: dark)
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility measured daily

VEHICLE CONTROL PERFORMED: n/a

RANGE-FINDING STUDY
- Test concentrations: 0.32, 1.0, 3.2 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes (20 % and 100 % immobilisation observed in the two highest concentrations, respectively. No immobilisation was observed in the lowest 3 concentrations).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate - study conducted on 03 June 2016.

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

7.85 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loaded test item

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4.94 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loaded test item

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: no
- Observations on body length and weight: not measured
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: yes
- Limit test: no
- Dose-response test: yes
- ECx: 24 h EC50 = 1.04 mg/L
- Other: satisfies guideline criteria of 0.6 - 2.1 mg/L.

Reported statistics and error estimates:

48 h EL50 95 % confidence limits = 4.37 - 5.58 mg/L

Table 1       Concentration of the test substance in the test water (limonene = 23.3 % of the test item mixture)

Nominal concentration (mg test item/L)	Expected nominal concentration in limonene (mg limonene/L)	Start (t = 0 h)		t = 24 h expired		t = 24 h fresh		End (t = 48 h)		Geometric mean measured concentrations
		Measured concentrations (mg/L)	% of nominal	Measured concentrations (mg/L)	% of nominal	Measured concentrations (mg/L)	% of nominal	Measured concentrations (mg/L)	% of nominal	mg/L	%
Control	0	< LOD	n/a	< LOD	n/a	< LOD	n/a	< LOD	n/a	< LOD	n/a
2.0	0.47	0.38	81	0.35	74	0.35	74	0.32	68	0.35	74
3.0	0.70	0.51	73	0.45	64	0.50	71	0.53	76	0.50	71
4.5	1.05	0.69	66	0.60	57	0.62	59	0.70	67	0.65	62
6.8	1.58	1.17	74	0.90	57	0.91	58	0.99	63	0.99	63
10	2.33	1.33	57	120	52	1.16	50	1.16	50	1.21	52

< LOD: below the limit of detection (0.08 mg/L)

% of nominal: percent of expected nominal concentration in limonene

Table 2       Acute immobilisation of Daphnids after 24 and 48 h in the main test

Nominal concentration* (mg test item/L)	Rep.	Number of daphnids exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Control	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
2.0	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
3.0	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
4.5	1	5	0	20	0	55
	2	5	0		2
	3	5	2		5
	4	5	2		4
6.8	1	5	1	40	3	75
	2	5	2		4
	3	5	2		4
	4	5	3		4
10	1	5	4	65	5	100
	2	5	5		5
	3	5	1		5
	4	5	3		5

* WAF prepared at given loading rate

 

 Table 3       pH values during the main test

		Nominal Concentration* (mg test item/L)
		Control	2.0	3.0	4.5	6.8	10
Start t =0		8.10	8.13	8.14	8.16	8.16	8.16
t = 24	Expired	8.09	8.10	8.11	8.09	8.10	8.12
	Fresh	8.10	8.08	8.12	8.10	8.09	8.11
End t = 48		8.12	8.10	8.12	8.08	8.14	8.10

* WAF prepared at given loading rate

 

 Table 4       Dissolved oxygen concentrations (mg/L) during the main test

		Nominal Concentration* (mg test item/L)
		Control	2.0	3.0	4.5	6.8	10
Start t =0		8.43	8.35	8.40	8.40	8.47	8.41
t = 24	Expired	8.33	8.51	8.61	8.60	8.69	8.57
	Fresh	8.51	8.52	8.54	8.54	8.50	8.50
End t = 48		8.69	8.50	8.49	8.51	8.48	8.52

* WAF prepared at given loading rate

All values correspond to values >90 % of the air-saturation value.

Validity criteria fulfilled:

yes

Conclusions:

The 48h EL50 of the test item to Daphnia magna was 4.94 mg/L (95% CI: 4.37 - 5.58).

Executive summary:

OECD 202 (2016) - The 48–hr-acute toxicity of the Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT) to Daphnia magna was studied under semi-static conditions.  Daphnids were exposed to a media control, and test chemical at (nominal) loading rates of 2.0, 3.0, 4.5, 6.8 and 10 mg/L} for 48 hr. Mortality/immobilization were observed daily.  The 48-h hour EL50, based on the nominal loading rate, was 4.94 mg a.i./L (95 % confidence limits = 4.37 - 5.58 mg/L).  The 48 -h NOEL based on mortality/immobilization was 3.0 mg/L.

Based on the results of this study, the Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT) would be classifed as Acute Category II to Daphnia magna in accordance with United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS)

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Results Synopsis;

Test Organism Age: < 24 h old

Test Type: Semi-static with 24 h renewals

EL50: 4.94 mg/L (95% C.I. : 4.37 to 5.58 mg/L)

NOEL: 3.0 mg/L

Description of key information

Constituent 1 ((4R)-4-isopropenyl-1-methylcyclohexene) 48h EC50 = 0.62 mg/L, REACH guidance on QSARs R.6, OECD Guideline 202 (Acute Daphnia sp. Immobilisation Test), with modifications from OECD Series on Testing and Assessment no. 23 on ecotoxicity testing of difficult substances (Anon., 2017).

A supporting study on the acute toxicity of the substance to Daphnia magna was also conducted in accordance with OECD 202 (Anon. 2016). The EL₅₀ was determined following 48h exposure of daphnids to water accommodated fractions of the test item, thus creating the conditions under which the key (Q)SAR predictions were made. The resulting 48h EL₅₀ was estimated as 4.94 mg/L (95 % CI: 4.37 - 5.58). The results of this study support the magnitude of the key effect levels generated by each (Q)SAR study for Daphnia sp., providing further evidence of their relevance and reliability for these endpoints.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.62 mg/L

Additional information

Short-term toxicity to Daphnia endpoints and expected concentrations of each constituent in the mixture

Constituent Name	48h-LC50^a (mg/L)	Concentration in the WAF (mg/L)
(4R)-4-isopropenyl-1-methylcyclohexene (limonene)	0.62	1.6
(4R)-4-(2-methoxy-2-propanyl)-1-methylcyclohexene (ether MT)	10	3.2
1-methyl-4-(2-propanylidene)cyclohexene (terpinolene)	0.73	0.53
cis-1-methoxy-1-methyl-4-(1-propen-2-yl)cyclohexane (cis-1-methoxy-8-pmenthene)	9.8	0.27
2-[(1S)-4-methyl-3-cyclohexen-1-yl]-2-propanol (α-terpineol)	61	0.18
trans-1-methoxy-1-methyl-4-(1-propen-2yl)cyclohexane (trans-1-methoxy-8-p-menthene)	9.8	0.09
1-methoxy-1-methyl-4-(2-propanylidene)cyclohexane (1-methoxy-4-p-menthene)	12	0.16
cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane (cis-1,8-dimethoxy-p-menthane)	163	0.51
trans-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane (trans-1,8-dimethoxyp-menthane)	163	0.08
7-methyl-3-methylene-1,6-octadiene (myrcene)	0.44	0.02
1-methyl-4-(2-propanyl)-1,3-cyclohexadiene (α-terpinene)	0.54	0.03
1-methyl-4-(2-propanyl)-1,4-cyclohexadiene (γ-terpinene)	0.73	0.03

^aindividual reports for each constituent presented in Section 6.1.3

Constituent 1 ((4R)-4-isopropenyl-1-methylcyclohexene) 48h EC50 = 0.62 mg/L is used for chemical safety assessment.