Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

Endpoint summary

Description of key information

Long-term earthworm information for Cyclabute using read across from Verdox (tested in OECD TG 222): EC10 is 45.1 mg/kg soil dw

Long-term plant test for Cyclabute using read across from Verdox (tested in OECD TG 208): EC10: 44 mg/kg soil dw

Long-term micro-organisms for Cyclabute using read across from Verdox (tested in OECD TG 216): NOEC: 100 mgkg soil dw

Additional information

The long-term terrestrial toxicity information for Cyclabute is derived from Verdox. First the experimental information from these terrestrial toxicity tests with Verdox will be presented and thereafter the read across justification.

Verdox and its long-term toxicity to earthworms

The long-term toxicity of the substance for earthworm Eisenia foetida was tested in OECD TG 222. The test material was dissolved in equal amounts of acetone, mixed with the quartz sand and allowed to slowly evaporate and mixed with the standard soil containing 10% Sphagnum peat, 20% kaolinite clay, approximately 70% fine quartz-sand (grain size 0.05 -0.2 mm) and 0.4% calcium carbonate to adjust to pH 6.0±0.5. Adult worms were exposed to nominal concentrations in soil of 5, 15.8, 50, 158.1 and 500 mg/kg dw soil. Weights of the adult worms ranged between 322 and 596 mg. The worms were fed weekly with finely ground cattle manure. Adult worms were removed after 4 weeks of exposure, counted and weighed. The remaining offspring remained in the test containers for another four weeks. Nominal concentrations are used to present the effects. No worms survived at 500 mg/kg soil dw. The NOEC for mortality and growth was 158.1 mg/kg dw. The EC10 for reproduction was 45.1 mg/kg dw soil (95% confidence interval is 21-65 mg/kg soil dw).

Verdox and its long-term toxicity to plants

In a terrestrial plant test according to OECD TG 208 under GLP, 6 plant species were tested for seedling emergence, growth and phytotoxic effects (chlorosis, necrosis and abnormal growth). The soil is a sandy loam and was sterilised before use. All particles were < 0.2cm. The organic matter content is 0.67 +/-0.03% and the pH was 5.9 +/-0.6. The substance was mixed with quarts sand and thereafter mixed into the soil. The following dicotyles were used: Brassica napus, Glycine max, Solanum lycopersicum and Cucumis sativus. The following monocotyles were use: Avena sativa and Allium cepa. Doses were selected based on a dose range finding study and were 12.3, 37, 111, 333 and 1000 mg/kg soil dw for most species. Two additional concentrations were used for Brassica napus and Glycine max: 1.37 and 4.12 mg/kg dw soil. One additional concentration was used for Solanum lycopersicum: 4.12 mg/kg dw soil. The concentration in the stock solution was verified but actual measures in the soil were not performed. Untreated seeds were sewn in pots and plants were grown for 14 days, except Allium cepa which was grown for 21 days. Fresh weight of the leaves of plants was the more sensitive endpoint compared to germination, phytotoxicity and mortality. The EC10 of all six plant species was averaged as the number of species =>5. The overall EC10 on leaf fresh weight being the most sensitive parameter resulted in 44 mg/kg soil dw based on nominal values and being the average of 6 plant species based on nominal values.

Verdox and its long-term toxicity to micro-organisms

The toxicity of Verdox for soil microflora was tested according to OECD TG 216 using nitrogen transformation in soil. The concentrations tested were 0, 10, 32, 100, 320 and 1000 mg/kg dw soil. The reference substance was sodium chloride. The soil used was abiologically active agricultural soil: Silty sand. The effect parameter was NO3-nitrogen production after 28 days exposure (soil nitrogen transformation). Nominal concentrations were used. No statistically significant effects of the test item on nitrate content in soil were observed at day 28 up to a concentration of 100 mg Verdox/kg soil dw by Williams Multiple t-test Procedure. For concentrations of 320 mg and 1000 mg Verdox/kg soil dw, the deviations to control were significant (Williams Multiple t-test Procedure). At day 28 differences in nitrogen production to the control were -30.25%, 36.26%, 35.90% for 10-100 mg/kg soil, not significantly and not concentration related. At 320 and 1000 mg/kg dw the reduction was -99.64% and -73.94% for 320 mg and 1000 mg Verdox /kg soil dw significantly though no clear dose relation. The NOEC was determined to be 100 mg/kg soil dw based on nominal values but an EC10 could not be calculated.

Cyclabute and its terrestrial toxicity using read across from Verdox (Cas no 20298-69-5)

Introduction and hypothesis for the analogue approach

Cyclabute is an isobutyl ester attached to a tricyclodecenyl fused ring backbone. For this substance no terrestrial toxicity information is available. In accordance with Article 13 of REACH, lacking information can be generated by means of applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the terrestrial toxicity Cyclabute the analogue approach is selected because for one closely related analogue reliable soil toxicity information is available.

Hypothesis: Cyclabute has the same terrestrial toxicity compared to Verdox based on similarity in chemical structure, bioavailability and mode of action.

Available experimental information: For Verdox long-term aquatic is available. Experimental information is present for Daphnia and algae, while for fish the long-term toxicity is derived from the BCF study.  Long-term terrestrial toxicity information for Verdox is available according to current guidelines and receives Kl. 1.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemicals are shown in the data matrix below. Physico-chemical properties thought relevant for aquatic and terrestrial toxicity are listed in the data matrix (see Data matrix).

Purity and impurities

Cyclabute is a reaction mass with a purity between 90-100%. The substance contains two key constituents, which are very similar. The double bond in the right ring (see Data matrix) can be at the 5-yl or the 6-yl position. The impurities are all below < 10%.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation.

Structural similarities and differences: Cyclabute and Verdox have an unreactive ester bond attached to a hydrocarbon backbone. These backbones have the same number of carbons.  Cyclabute has an isobutyl-ester while Verdox has an acetic ester. The ester-alkyl chain is therefore two carbons longer compared to Verdox.

Bioavailability and biodegradation: Cyclabute (target) and Verdox have similar bioavailability based on the similarity in chemical structure and physico-chemical properties. The molecular weight of the target chemical is 220, whereas Verdox it is 198. They both have low melting points. The water solubility (16 and 9.7 mg/l), log Kow (5.1 and 4.75) and Koc (3.5 and 3.1), respectively. The vapour pressure of Cyclabute is slightly lower compared to Verdox 0.61 and 9.7, respectively). Cyclabute and Verdox are both biodegraded into a similar hydrocarbon secondary alcohol and the respective acid:  Iso-butanoic-acid for Cyclabute, acetic acid for Verdox. These acids are readily biodegradable, while the respective alcohols are somewhat more stable. This shows that the fate in soil will also be similar.

Mode of action: Cyclabute and Verdox have both the ester as the functional group and on that bases have a similar mode of action.

Uncertainty of the prediction: In view of the reasoning above there are no uncertainties. The slight difference in measured log Kow is expected to be due to experimental variability. The calculated log Kow is similar and also the measured Kocs are very similar. Though Cyclabute is slightly less volatile compared to Verdox this has not affected the results. It can be seen that the adsorption to soil and the concentrations are the key drivers for toxicity.  In addition, when converting the PNEC soil of Verdox towards Cyclabute using the molecular weight and log Kow the result remains almost the same:

44 mg Verdox/kg soil dw /198 (Verdox)*220 (Cyclabute) MW * 4.75/5.1 = 45.5 mg/kg soil dw.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented

Conclusions per endpoint for terrestrial hazard identification

For Cyclabute no terrestrial toxicity data are available. For the analogue Verdox such data are available which can be used for read across. This read across information is applicable for risk assessment and is presented here with adequate and reliable documentation. For the analogue Verdox long-term terrestrial toxicity is available for 3 terrestrial species resulting in a PNEC soil of 4.4 mg/kg soil dwt, which can be used for read across to Cyclabute.

Final conclusion: Cyclabute has a PNEC soil of 4.4 mg/kg dwt soil.

Data matrix of Cyclabute for terrestrial toxicity using read across from Verdox

Common names	Cyclabute (Target)	Verdox (Source)
Chemical structures
Cas no of the main isomer Cas no of the generic	67634-20-2 (5-yl) 93941-73-2	20298-69-5
EINECS no	916-331-7	243-718-1
REACH registered	2018	2013
Empirical formula	C14H20O2	C12H22O2
Physico-chemical data
Molecular weight	220	198
Physical state	liquid	Liquid
Melting point (°C)	< -20	29.8
Boiling point (°C)	275	232
Vapour pressure (Pa) (measured)	0.61	9.72
Water solubility (mg/l) (measured)	16	10
Log Kow (measured)	5.1	4.75
Log Kow (Episuite)	3.76	4.42
Fate
Ready biodegradation %	22	43
Log Koc	3.5	3.12
Aquatic toxicity
PNEC aquatic toxicity mg/l	0.0163 (based on all acute and long-term aq. Tox)	0.057 (experimental algae and Daphnia, fish predicted)
Terrestrial toxicity
PNEC based on terrestrial data mg/kg dwt		4.4
Earthworm EC10 or NOEC in mg/kg soil dw	45.1 based on read across from Verdox*	45.1 (OECD TG 222)
Plants (EC10 or NOEC, average of 6 species) mg/kg soil dw	44 based on read across from Verdox*	44 (OECD TG 208)
Micro-organisms ( NOEC) mg/kg  soil dw	100 based on read across from Verdox*	100 (OECD TG 216)

*Conversion is not considered necessary in view of the minimal changes and Verdox presenting a conservative approach. The lowest EC10 of Verdox is for plants: 44 mg/kg soil dw Converting this toward Cyclabute with a slightly higher molecular weight and higher log Kow this results in

44 mg/kg soil dw /198*220 MW * 4.75/5.1 = 45.5 mg/kg soil dw.