3.2.2.1. Safety for the target species
Tolerance studies in the target species and/or toxicological studies in laboratory animals made with the essential oil under application were not submitted.
In the absence of these data, the approach to the safety assessment of a mixture whose individual components are known is based on the safety assessment of each individual component (component‐based approach). This approach requires that the mixture is sufficiently characterised and that the individual components can be grouped into assessment groups, based on structural and metabolic similarity. The combined toxicity can be predicted using the dose addition assumption within an assessment group, taking into account the relative toxic potency of each component (EFSA Scientific Committee, 2019a).
As the additive under assessment is a fully defined mixture (the identified components represent > 99.8% of the % GC area, see Section 3.2.1), the FEEDAP Panel applied a component‐based approach to assess the safety for target species of the essential oil.
Based on considerations related to structural and metabolic similarities, the components were allocated to nine assessment groups, corresponding to the chemical groups (CGs) 6, 8, 16, 32 and 31, as defined in Annex I of Regulation (EC) No 1565/2000. For CG 31 (‘aliphatic and aromatic hydrocarbons’), subassessment groups as defined in Flavouring Group Evaluation 25 (FGE.25) and FGE.78 were established (EFSA CEF Panel, 2015a,b). The allocation of the components to the (sub‐)assessment groups is shown in Table 5 and in the corresponding footnote.
For each component in the assessment group, exposure of target animals was estimated considering the use levels in feed, the percentage of the component in the oil and the default values for feed intake according to the guidance on the safety of feed additives for target species (EFSA FEEDAP Panel, 2017b). Default values on body weight are used to express exposure in terms of mg/kg bw per day. The intake levels of the individual components calculated for chickens for fattening, the species with the highest ratio of feed intake/body weight per day, are shown in Table 5.
For hazard characterisation, each component of an assessment group was first assigned to the structural class according to Cramer classification (Cramer et al., 1978). For some components in the assessment group, toxicological data were available to derive no observed adverse effect level (NOAEL) values. Structural and metabolic similarity among the components in the assessment groups were assessed to explore the application of read‐across allowing extrapolation from a known NOAEL of a component of an assessment group to the other components of the group with no available NOAEL or, if sufficient evidence were available for members of a (sub‐)assessment group, to derive a (sub‐)assessment group NOAEL.
Toxicological data of subchronic studies, from which NOAEL values could be derived, were available for linalool [02.013] and terpineol24 [02.230] in CG 6 (EFSA FEEDAP Panel, 2012c), 1,8‐cineole in CG 16 (EFSA FEEDAP Panel, 2012b, 2021), myrcene [01.008], d‐limonene [01.045], p‐cymene [01.002] and β‐caryophyllene [01.007] in CG 31 (EFSA FEEDAP Panel, 2015, 2016a), and β‐caryophyllene epoxide in CG 32 (EFSA CEF Panel, 2014).
Considering the structural and metabolic similarities, for the subgroup of terpinyl derivatives in CG 6, i.e. α‐terpineol [02.014] and 4‐terpinenol [02.072], the reference point was selected based on the NOAEL of 250 mg/kg bw per day available for terpineol [02.230] and d‐limonene [01.045].
Similarly, the NOAELs of 44, 250, 154 and 222 mg/kg bw per day for the representative compounds of CG 31, myrcene [01.008], d‐limonene [01.045], 1‐isopropyl‐4‐benzene [01.002] and β‐caryophyllene [01.007] were applied, respectively, using read‐across to the compounds within subassessment group II (trans‐β‐farnesene), group III (γ‐terpinene, terpinolene, α‐terpinene, β‐elemene, α‐phellandrene and β‐sesquiphellandrene), group IVe (m‐cymene) and group V (α‐pinene, sabinene, β‐pinene, δ‐cadinene, α‐thujene, camphene, α‐cubebene, longifolene, α‐copaene, β‐cubebene, γ‐cadinene, α‐selinene, α‐cadinene, β‐selinene, δ‐3‐carene, cubebene, tricyclene, selina‐3,7(11)‐diene, α‐fenchene, δ‐amorphene, α‐longipinene, zonarene, β‐copaene and selina‐3,6‐diene)25 (EFSA CEF Panel, 2015a,b).
For the remaining compounds,26 NOAEL values were not available and read‐across was not possible. Therefore, the TTC approach was applied (EFSA FEEDAP Panel, 2017b).
As the result of the hazard characterisation, a reference point was identified for each component in the assessment group based on the toxicity data available (NOAEL from in vivo toxicity study or read across) or from the 5th percentile of the distribution of NOAELs of the corresponding Cramer Class (i.e. 3, 0.91 and 0.15 mg/kg bw per day, respectively, for Cramer Class I, II and III compounds, Munro et al., 1996). Reference points selected for each compound are shown in Table 5.
For risk characterisation, the margin of exposure (MOE) was calculated for each component as the ratio between the reference point and the exposure. For each assessment group, the combined (total) margin of exposure (MOET) was calculated as the reciprocal of the sum of the reciprocals of the MOE of the individual substances (EFSA Scientific Committee, 2019a,b). A MOET > 100 allowed for interspecies differences and intraspecies variability (as in the default 10 × 10 uncertainty factor). The compounds resulting individually in an MOE > 50,000 were not further considered in the assessment group as their contribution to the MOE(T) is negligible. They are listed in the footnote.27
The approach to the safety assessment of juniper oil for the target species is summarised in Table 5. The calculations were done for chickens for fattening, the species with the highest ratio of feed intake/body weight and represent the worst‐case scenario at the use level of 10 mg/kg in feed.
As shown in Table 5, for all the assessment groups, the MOET was higher than 100, except for one group (CG 31, VI). From the lowest MOET of 39 for chickens for fattening, the MOET for the assessment group ‘macrocyclic non aromatic hydrocarbons’ (CG 31, VI) was calculated for the other target species considering the respective daily feed intake and conditions of use. The results are summarised in Table 6.
At the proposed use levels in complete feed, the MOET exceeds the value of 100 for laying hens, veal calves, cattle for fattening and ornamental fish, but is below the value of 100 for the other species. For the other species, the maximum safe use levels in feed were calculated in order to ensure a MOET ≥ 100. Because glucuronidation is an important metabolic reaction to facilitate the excretion of the components of the essential oil, the use of juniper oil as additive in cat feed needs a wider margin of exposure. Considering that cats have a low capacity for glucuronidation (Court and Greenblatt, 1997; Lautz et al., 2021), a MOET of 500 is considered adequate. The maximum safe levels in feed are shown in Table 6.