Registration Dossier

Administrative data

Hazard for aquatic organisms

Freshwater

Hazard assessment conclusion:
PNEC aqua (freshwater)
PNEC value:
5.4 µg/L
Assessment factor:
50
Extrapolation method:
assessment factor
PNEC freshwater (intermittent releases):
5.77 µg/L

Marine water

Hazard assessment conclusion:
PNEC aqua (marine water)
PNEC value:
0.54 µg/L
Assessment factor:
500
Extrapolation method:
assessment factor

STP

Hazard assessment conclusion:
PNEC STP
PNEC value:
2.1 mg/L
Assessment factor:
10
Extrapolation method:
assessment factor

Sediment (freshwater)

Hazard assessment conclusion:
PNEC sediment (freshwater)
PNEC value:
1.3 mg/kg sediment dw
Extrapolation method:
equilibrium partitioning method

Sediment (marine water)

Hazard assessment conclusion:
PNEC sediment (marine water)
PNEC value:
0.13 mg/kg sediment dw
Extrapolation method:
equilibrium partitioning method

Hazard for air

Air

Hazard assessment conclusion:
no hazard identified

Hazard for terrestrial organisms

Soil

Hazard assessment conclusion:
PNEC soil
PNEC value:
0.261 mg/kg soil dw
Extrapolation method:
equilibrium partitioning method

Hazard for predators

Secondary poisoning

Hazard assessment conclusion:
no potential to cause toxic effects if accumulated (in higher organisms) via the food chain

Additional information

Orange oil is a complex substance consisting of a mixture of poorly soluble constituents. Therefore the aquatic toxicity was determined in tests with Water Accomodated Fractions. These tests were performed with algae, daphnids and fish. The key study was one of the two Daphnia studies with the lowest result: EL50 = 1.1 mg Orange oil/L (loading rate). Results of WAF studies, however, cannot be used for the risk assessment as (1) it is not possible to quantify the (integrated) environmental exposure for all constituents together and (2) it is not possible to derive a meaningful PNEC based on WAF test results.

 

A DPD+ analysis (ref. 1) for Orange oil identified d-limonene to represent the dominant risk to the environment. All constituents of the complex substance Orange Oil have the same working mechanism (Narcosis and Polar narcosis) and there are no other constituents that are highly toxic to the environment. It is concluded that for the environmental risk assessment, d-Limonene is representative for the other constituents.

Therefore, the environmentl exposure assessment will be carried out using limonene as the lead constituent and also the PNEC for d-limonene will be used for the risk characterisation.

(Ref. 1): REACH: Exposure scenarios for preparations. Methodology for the identification of substances that represent the dominant risks to human health and/or the environment and the drivers for risk management measures, CEFIC, June 09: final for publication

Conclusion on classification

Orange oil is a complex substance for which WAF testing is the suitable approach. It was shown that the influence of minor constituents is essential for the determination of its aquatic toxicity. Orange oil is to be classified as Aquatic Chronic Category 2.

 

1. Introduction

 

Orange oil is a substance as defined by REACH art. 3.1 and for Registration it is identified as a Natural Complex Substance,category UVCB. For data gathering and hazard classification of substances, the CLP Regulation (EC) 1272/2008 prescribes a tiered approach: tests are to be carried out with the substance or mixture in the form in which it is marketed and used. Valid data on a substance or mixture prevail for classification and labelling. Only in the absence of data on the substance or mixture, valid data of the constituents should be taken following the principles for the classification of mixtures (art. 6.5 of CLP).

 

For the substance Orange oil “adequate, reliable and scientifically valid” data exist for algae, Daphnia and fish to justify the assigned environmental hazard classification as Aquatic Chronic Category 2. This is explained in detail in the attachment to this IUCLID section 6.0 (document “Environmental Classification of Orange Oil).

 

Orange oil is a Natural Complex Substance (NCS) with D-Limonene as a significant constituent, but it also contains a range of other constituents of highly different water solubility in variable concentrations, ranging from < 1 up to 1560 mg/L. The constituents in the NCS interact with each other and influence each other’s solubility in water, so their concentration ratios in water differ from those in the substance Orange oil. Upon addition to water, equilibrium will be reached between the dissolved and undissolved fractions which will be characteristic of the loading rate of the substance in water. The presence and interaction of the constituents in the water phase determine the aquatic toxicity of Orange oil. Therefore the short-term aquatic toxicity of Orange oil was tested as a WAF (Water Accommodated Fraction) as recommended for test items with partially soluble constituents (OECD Series on testing and assessment No 23, OECD 2000). The WAF tests delivered valid data which are used for the classification of Orange oil.

 

According to the tiered approach in the CLP Regulation, in the absence of valid test results for the substance Orange oil, a summation method for the Classification and Labelling of mixtures could be applied based on a theoretical extrapolation from the concentrations of the constituents in Orange oil. However, by ignoring the difference in water solubility and the mutual interaction between constituents, the aquatic toxicity of Orange oil is overestimated. Recently developed QSARs that take into account the difference in water solubility and mutual interaction of the constituent’s physico-chemical behaviour support the use of the WAF approach for Orange oil.

 

2. Regulatory background

Under REACH, Orange oil is a substance([1]) and for the purpose of registration it is considered as a UVCB sub-type 3. According to the ECHA Guidance on the Application of the CLP Criteria (V. 4.0, Nov 2013), the aquatic toxicity of poorly soluble UVCB substances can be assessed using water accommodated fractions (WAF) where the toxicity cutoff is related to the loading rate (CLP Guidance, § 1.2.3.3, § 4.1.3.2.2; ECHA Guidance on Information requirements and chemical safety assessment R.7b, p. 76-77). The median effective loading level from the WAF is used directly to determine the classification. WAF testing is justified for substances if solubility and other physico-chemical properties of the constituents are different, and the UVCB substance cannot be dissolved into a homogeneous solution (CLP Guidance, OECD Series on testing and assessment No 23, OECD 2000).

For the use of data from WAF studies on Orange oil in the context of CLP, the following articles in the CLP Regulation are relevant: 

 

Article 5.1 of the CLP Regulation (on the examination of available information on substances) states that:

The information shall relate to the forms or physical states in which the
substance is placed on the market and in which it can reasonably be
expected to be used.

 

This information shall be examined to ascertain whether it is adequate, reliable and scientifically valid for the purpose of classification.

 

Article 8 (on generating new information for substances and mixtures), article 8.6 also stipulates that:

Tests that are carried out for the purposes of this Regulation shall
be carried out on the substance or on the mixture in the form(s) or
physical state(s) in which the substance or mixture is placed on the
market and in which it can reasonably be expected to be used.

 

Thus the aquatic toxicity data shall be available for the form in which the substance is placed on the market and in which it will be used. The aquatic toxicity studies using WAFs prepared from Orange oil meet the validity requirements.

 

This approach is also explicitly reflected in the tiered approach for mixtures in Annex I, part 4.1.3.2 and in the accompanying figure 4.1.2 on the CLP Regulation.

 

The constituent D-Limonene has been registered as a substance under REACH (CAS no 5989-27-5) and it has a harmonised classification for the environment (H400, H410, M=1[2]). If Orange oil were taken as a theoretical mixture of Limonene and other constituents, the following articles of the CLP regulation could be considered as well:

 

Article 11 (Cutoff values): this specifies the cutoff values for relevant constituents for which information shall be taken into account when classifying mixtures in the absence of valid test data on the mixture. This article is secondary to Articles 5 or 6 which give precedence to actual test data: available valid information for the substance or mixture as a whole shall be used. This is also reflected in the tiered approach as outlined in Annex I, part 4.1.3.2 and the abovementioned figure.

 

Article 16.2 (Harmonized classification) states that a harmonised classification of a substance cannot be neglected. Contrary to the substance Limonene with its harmonised classification due to its inclusion in Part 3 of Annex VI, the substance Orange oil does not have a harmonised classification.

Although Limonene forms a major constituent in the NCS Orange oil, it is not the same substance and reliable data obtained on the substance Orange oil as such are the basis for its hazard assessment [3].

 

In conclusion, regardless of Orange oil being a substance or being regarded as a theoretical mixture, the aquatic toxicity data for the form in which it is placed on the market and in which it will be used, shall drive the hazard classification if such data are available. As the aquatic toxicity studies using WAFs prepared from Orange oil meet the validity requirements (see next section), the WAF study results on Orange oil shall be used in the evaluation for the hazard classification.

 

3. Test data and classification of Orange oil

 

For the classification of the NCS Orange oil, the following results from WAF studies are available:

 

Results of WAF studies on the substance Orange oil

Orange oil quality

Algae, mg/L

Daphnia, mg/L

Fish, mg/L

Reference / Remarks

Orange terpenes

Limonene 93-95% W/W

72h-ErL50 ~150 #,

Rel. 2

48h-EL50 = 8.6,

Rel. 2

 

Lebertz 2007

Daily refreshments, closed system for Daphnia

Analysis of total hydrocarbons by IR spectroscopy

Orange terpenes

Limonene 98.39 area%

 

 

96h-EL50 = 5.65,

Rel. 1

Lebertz 2011

Daily refreshments, closed system

Analysis of total hydrocarbon concentration by IR spectroscopy

Orange oil cold pressed 1 -fold

Limonene 95.1 %

 

48h-EL50 = 1.1, 

Rel. 1

 

Vinken and Wydra 2007 Closed test system

Analysis of Limonene concentration by GC/MS

# The value of circa 150 mg/L for algae is far above the water solubility of many of the constituents. The high value may be related to loss due to volatility of constituents during the preparation of the WAF. During the test the concentration of total hydrocarbons remained above 80% of the initial concentration. There is no indication that algae are more sensitive than daphnids and fish.

The following approach for classification has been taken, in line with the criteria in the CLP Regulation (EC) No 1272/2008, Annex I part 4, Table 4.1.0:

 

1.      The classification for aquatic acute toxicity is based on available WAF studies that are adequate, reliable and scientifically valid (Reliability 1 or 2). The short-term EL50 are > 1 mg/l for algae, Daphnia and fish. This implies that Orange oil is not classified for acute aquatic hazards under CLP Regulation (EC) No 1272/2008. The robust study summaries of these studies are included in the current technical dossier.

2.      The classification for aquatic chronic toxicity is based on the same WAF studies. Orange oil is rapidly biodegradable but log Kow > 4 for 83% of the typical constituent composition. The short-term EL50 values for the most sensitive organisms, Daphnia and fish, are between 1 and 10 mg/L. This leads to classification as Aquatic Chronic Category 2 (according to CLP Regulation (EC) No 1272/2008 as amended by Commission Regulation (EU) 286/2011, table 4.1.0 (b)(iii)). 

In conclusion, based on valid aquatic toxicity studies performed for the complex substance Orange oil, Orange oil is not classified for Aquatic Acute toxicity. Orange Oil is to be classified as Aquatic Chronic Category 2.   

4. Observations on the solubility in tests with Limonene and Orange oil

 

Orange oil typically consists of 80% Limonene and other constituents with a wide range of water solubilities. Orange oil and Limonene are both registered under REACH as individual substances. The difference between Limonene and Orange oil is obvious if only from the smell. As some varieties of Orange oil contain a high Limonene content, a comparison of Orange oil with Limonene is inevitable.

 

Behaviour of Limonene

When the Daphnia toxicity study on Orange oil was carried out by IBACON (Vinken and Wydra 2007), a study with the substance Limonene was performed in the same period in exactly the same way (see REACH dossier for Limonene). Water Accommodated Fractions were prepared for Orange oil as well as for Limonene and the test design and method for chemical analyses were identical. Limonene concentrations were determined in freshly prepared aqueous medium as well as after 48 h. This coincidence of studies gives the opportunity to assess the differences for both substances in the two identical tests. Assuming that Limonene behaves independently of the other constituents in the solution, Limonene should behave exactly the same in the medium with Orange oil as in the medium with the substance Limonene and the deviations between the measured and the nominal Limonene concentration should be identical. The analytical and effects data presented in the IBACON reports have been analysed to test this hypothesis. Details are given in the attachment of this IUCLID section.

 

The analytical results for the replicate samples per time (analytical replicates) match very well. For the test substance Limonene, the Limonene concentrations obtained in the WAF preparation are overall lower than those from Orange oil. In the highest concentrations of the freshly prepared WAFs of Orange oil, the Limonene concentration was consistently higher than in the ones for the substance Limonene. As this is observed in all 5 test levels this difference cannot be attributed to coincidence. The decrease in the Limonene concentrations over time is also higher in the Limonene WAF (minus 20%) than in the Orange oil WAF (minus 8%).

 

In conclusion, in the Orange oil WAF we observe:

1.   A significantly higher solubility of Limonene (initially 136 to 163%);

2.   A significantly higher stability of the concentration of Limonene over time.

 

This difference may be attributed to the presence of other constituents from Orange oil that mutually influences the behaviour of all constituents in the WAF. This aspect is well recognized following thermodynamic principles of mixtures (see Section 5).

Toxic effects

If Limonene behaved independently of the circumstances, the higher concentrations of Limonene found in the Orange oil WAF would lead to higher toxicity than in the pure Limonene WAFs. However, the contrary is observed indicating that the other constituents present in the WAF of Orange oil influence the toxicity to Daphnia (i.e. the test data obtained from the testing of the Orange oil WAFs apply to the complex substance as an entity).

 

These observations support the conclusion that Orange oil is not identical to the substance Limonene. Orange oil is a complex substance and a test using a WAF is scientifically justified.

5. QSAR support for using WAF test results 

High Accuracy QSARs for investigating complex substance toxicity (iSafeRat®) were used to predict the toxicity of three Orange oil qualities in WAF tests.The acute aquatic toxicity of multi-constituent substances is assessed by a multi-step calculation method based on the chemical activity of the constituents in water. A thermodynamic approach calculates the influence of each constituent on their respective solubility allowing the concentration of each constituent within the complex substance to be resolved (i.e. providing the “loading rate”). In a next step, the non-bioavailable fraction is removed to determine the true concentration of each constituent exerting toxicity. This concentration is calculated by the model in terms of a fraction of the chemical activity exerted by each constituent. Finally, the loading rate is adjusted until the value of the sum of activities of the bioavailable fractions is equal to the fraction-weighted average of toxic activity of each constituent. This can be seen as mechanistically following the same process which occurs in a WAF study.

The model has been validated using 12 GLP WAF studies following OECD Guidelines (4 on fish, 5 on daphnids and 3 on algae) in which the aqueous concentrations of several of the constituents and also Lethal or Effect loading rates (E/LL50) were determined. The predictions of the model were all within a factor of 2 of the study outcomes. Thus the predictions can be considered as accurate as the laboratory studies themselves. A full explanation is provided in the Attachment to this IUCLID section (document "Environmental Classification of Orange Oil").

 

The results of the WAF calculations by the QSAR-WAF calculation method iSafeRat® for the three Orange oil qualities are 2.8-3.1 mg/L for fish, 3.9 – 4.3 mg/L for Daphnia and 4.3 – 4.8 mg/L for algae. These results are close to those determined in WAF laboratory experiments (all >1 and vary between 1.1 and 8.6 mg/L) for daphnids and fish. The predicted sensitivity for algae is in the same range. The high empirical value of circa 150 mg/L for algae is somehow out of range but this seems to be related to loss of test material during preparation of the WAFs. However, there is no indication that algae are more sensitive than daphnids and fish.

 

Thus the QSAR-WAF calculation for Orange oil supports the experimental values in WAF toxicity tests for daphnids and fish.

6. Summary and conclusions

Orange oil is a complex substance for which WAF testing is the suitable approach. It was shown that the influence of minor constituents is essential for the determination of its aquatic toxicity. Orange oil is to be classified as Aquatic Chronic Category 2.

An analysis of the Regulatory requirements of the CLP regulation leads to the conclusion that regardless of Orange oil being a substance or being regarded as a theoretical mixture, the aquatic toxicity data for the form in which it is placed on the market and in which it will be used, shall drive the hazard classification if such data are available (tiered approach).

Aquatic toxicity studies using WAFs prepared from Orange oil are available and meet the validity requirements. Therefore, the WAF study results on Orange oil shall be used in the evaluation for the hazard classification.

The toxicity for aquatic organisms of the UVCB substance Orange oil was assessed in WAF tests. These studies for algae, Daphnia and fish are considered valid studies. Based on these valid aquatic toxicity studies and the criteria of the CLP Regulation, the substance Orange oil is not classified for Aquatic Acute toxicity. Orange Oil is to be classified as Aquatic Chronic Category 2.  

 

The UVCB substance Orange oil and the single substance Limonene were both tested in the same WAF test design in the same period in the same laboratory. This offers the opportunity to compare the fate of the constituent Limonene and the toxicity of the two substances.

It is concluded that the substance Orange oil, even with a content of 95% Limonene, does not behave as if it were the substance Limonene. The presence of other constituents in the complex substance Orange oil influences the behaviour and effect of the constituent Limonene. Therefore a test using a WAF is scientifically justified. The result based on the loading rate of Orange oil (48h-EL50 of 1.1 mg/L) for Daphnia as the most sensitive species is the key result to be used for classification.

 

The experimental results in WAF tests are supported by High Accuracy QSARs:the results determined by the QSAR-WAF calculation method (iSafeRat®) are of the same order of magnitude as those determined in WAF laboratory experiments (all >1 and ranging between 1.1 and 8.6 mg/L for daphnids and fish). Thus for Orange oil, the QSAR-WAF calculations support the toxicity values found experimentally.

Footnotes

[1]Definitions in REACH and CLP Regulations:‘substance’ means a chemical element and its compounds in the natural state or obtained by any manufacturing process, including any additive necessary to preserve its stability and any impurity deriving from the process used, but excluding any solvent which may be separated without affecting the stability of the substance or changing its composition; ‘mixture’ means a mixture or solution composed of two or more substances.

[2]  Currently the classification of Limonene is under revision (August 2015: ECHA website, CLH intentions). As a result of recent test data the chronic classification H410 is expected to become H411 (source EFEO and IFEAT).

[3]Contrary to the classification, for the environmental risk assessment of Orange oil, Limonene is taken as the lead constituent. Results from WAF studies cannot be used in the risk assessment, as it is not possible to evaluate the (integrated) environmental exposure for all constituents together.