[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Endpoint summary

Description of key information

In a 28-day repeated-dose oral toxicity study with Delta-damascone a systemic NOAEL of 85 mg/kg bw has been derived based on the observed hepatoxicity at the next dose level. In a 90-day oral study with its structural analogue Alpha-iso-methylionone, a NOAEL of 30 mg/kg bw/day was established based on increases in plasma levels of creatinine, total protein and cholesterol (indicators of liver effects), increases in absolute and relative weights of the kidneys and spleen, and microscopic changes in thyroid and bone marrow at the next dose level. At the highest dose level also liver effects were seen at the 500 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The information of the two available studies are considered reliable and sufficiently adequate for the present dossier.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study plan:

An extended OECD TG 422 with Delta Damascone will be performed and the final report is expected to be finished Q4 2022/Q1 2023. Once the study is available, the studies with the structural analogue will be replaced with the extended OECD 422 study using Delta Damascone. Until then data on Alpha-iso-methylionone is used for hazard assessment and DNEL derivation.

Current risk assessment

No sub-chronic or chronic studies were available for Delta-damascone. However, Article 13 of REACH states that, in case no appropriate animal studies are available for assessment, information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. A 90-days repeated dose toxicity study is available for a structural analogue of Delta-damascone: Alpha-iso-methylionone. First the executive summary of the key and supporting studies are presented and thereafter the read across justification.

Key: 90-day repeated dose toxicity study on Alpha-iso-methylionone

In a 90-day repeated dose toxicity study, performed according to OECD Guideline 408 and in accordance with GLP, the substance was administrated by oral gavage at dose levels of 500, 30 and 5 mg/kg bw/day for a period of 90 consecutive days to groups of 10 male and 10 female Sprague-Dawley rats. A control group of the same size was treated with the vehicle (corn oil) only. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no mortalities or adverse clinical signs noted. There were no treatment-related changes in the behavioural and functional performance parameters measured. There were no treatment-related changes in sensory reactivity, bodyweight development, food consumption, water consumption, ophthalmoscopy, haematology, and no macroscopic abnormalities were detected. Animals of either sex treated with 500 mg/kg/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol (p<0.01) compared to control animals. Males from this treatment group also showed a statistically significant increase in plasma albumin (p<0.01). No such effects were detected in animals of either sex treated with 30 or 5 mg/kg/day. Animals of either sex treated with 500 mg/kg/day showed a statistically significant increase in liver weight (p<0.01) both absolute and relative to terminal bodyweight. Males treated with 500 mg/kg/day also showed statistically significant increases in absolute and relative spleen weight (p<0.01) and in both sexes an increase in absolute and relative kidney weight (p<0.01) was observed. No such toxicologically significant effects were detected in animals of either sex treated with 30 or 5 mg/kg/day. Hepatocyte enlargement, centrilobular or generalised, was observed in relation to treatment for 4/10 (minimal) males and 9/10 (minimal) females treated with 500 mg/kg/day (p<0.05 for males; p<0.001 for females). Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature. A greater incidence of higher severity grades of globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/10 (minimal), 5/10 (slight), 1/10 (moderate) males treated with 500 mg/kg/day (p<0.01) or in 5/10 (minimal), 4/10 (slight) males treated with 30 mg/kg/day (p<0.05) but not at 5 mg/kg/day. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of alpha2-microglobulin in renal proximal tubular epithelial cells. This is a well documented effect, peculiar to the male rat, which occurs in response to treatment with certain hydrocarbons. Female rats and other species do not develop "hydrocarbon nephropathy" and for this reason the effect may not be indicative of hazard to human health. Two males treated with 500 mg/kg/day and one male treated with 30 mg/kg/day exhibited associated higher grades of tubular basophilia. A higher incidence of follicular cell hypertrophy was seen in relation to treatment for 7/10 (minimal) males treated with 500 mg/kg/day (p<0.01). A higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for 7/10 (minimal) and 3/1 (slight) males treated with 500 mg/kg/day (p<0.01).

The NOAEL for systemic toxicity has been set at 30 mg/kg bw/day, based on increases in plasma levels of creatinine, total protein and cholesterol (indicators of liver effects), increases in absolute and relative weights of the kidneys and spleen, and microscopic changes in thyroid and bone marrow at the next dose level.

Supporting: 28-day repeated dose toxicity study on Delta-damascone

In a study performed in compliance with the Japanese guideline '28 -day Repeated Dose Toxicity Study in Mammalian Species' (Concerning Testing Methods Relating to the New Chemical Substances) and GLP, the test substance was administered to five male and female Crl:CD(SD) rats per group by oral gavage as a dilution in corn oil at dose levels of 0, 21, 85 and 340 mg/kg/day. Additionally, the study included two recovery groups of 5 rats/sex each which were dosed for 28 days with the vehicle (control) or 340 mg/kg of test substance and then left untreated for 14 days. During the dosing period, general clinical observations, detailed clinical observations, function examinations, body weight measurements and food consumption measurements were performed. On the day after the last dosing, urinalyses, blood examinations and pathological examinations were performed after collecting urine and blood samples.

In the general clinical observations, decreased spontaneous locomotion in males and females of the 85 mg/kg groups and higher, incomplete eyelid opening in males of the 85 mg/kg group and higher and females of the 340 mg/kg group, lacrimation, reddish tear and staining hair in males and females of the 340 mg/kg groups, decreased respiratory rate, loss of hair and moist hair in males of the 340 mg/kg group and staining around external genitalia, staining lower abdomen and staining around anus in females of the 340 mg/kg group were observed. Salivation in males and females of the 85 mg/kg groups and higher and staining around nose and mouth, much food on the tray (increase of the amount of food dropped on tray by biting) and restlessness which meant increased chin rubbing or cage scratching in males and females of the 340 mg/kg groups were also observed. In the detailed clinical observations, staining hair in males and females in week 1, 2 and 4, salivation in males and females in week 4 and lacrimation in males in week 4 were observed in the 340 mg/kg groups. In the body weight, lower tendencies were found from day 8 to day 28 of the dosing period in males of the 340 mg/kg group. In the food consumption, lower tendencies on day 3 of the dosing period in males and females, statistically significant increases or a higher tendency from day 15 to day 28 of the dosing period in females and a higher tendency on day 28 of the dosing period in males were found in the 340 mg/kg groups. In the blood chemical examinations, statistically significant decreases of total protein and albumin in males of the 85 mg/kg group and higher, statistically significant increases of gamma-glutamyl transpeptidase in males and females of the 340 mg/kg groups, statistically significant increases of aspartate aminotransferase and total bilirubin, a higher tendency of alanine aminotransferase (ALT) and a lower tendency of triglyceride in males of the 340 mg/kg group and a statistically significant decrease of choline esterase (ChE) in females of the 340 mg/kg group were found. In the organ weight, statistically significant increases of relative weights of the liver were found in males and females of the 340 mg/kg groups. Statistically significant increases of relative weight of the kidneys were also found in males and females of the 340 mg/kg groups. In the gross necropsy, blackish region of mucosa in the glandular stomach in females of the 85 mg/kg group and higher and males of the 340 mg/kg group, enlargement of the liver in males and females of the 340 mg/kg groups and recessed region of mucosa in the forestomach in female of the 340 mg/kg group were observed. In the histopathological examinations, hypertrophy and prominent nucleoli of periportal hepatocytes in the liver in males of the 85 mg/kg group and higher and females of the 340 mg/kg group, focal necrosis of fundic mucosa in the glandular stomach in females of the 85 mg/kg group and higher and males of the 340 mg/kg group, spongiosis of squamous epithelium and oedema in lamina propria in limiting ridge and oedema in submucosal layer in the forestomach, and oedema in submucosal layer in the glandular stomach in males of the 340 mg/kg group and focal spongiosis of squamous epithelium in the forestomach in female of the 340 mg/kg group were observed. No abnormal changes were observed in the sensorimotor function examinations, urinalyses, haematological examinations and oestrous cycle stage. In the recovery group, most of changes observed in dosing period and at the end of dosing period disappeared, although much food on the tray on day 1 of the recovery period, statistically significant decreases of the body weight from day 1 to day 14 of the recovery period and a statistically significant increase of ALT at the end of recovery period in males and staining around external genitalia on day 1 of the recovery period, a statistically significant decrease of ChE and a statistically significant increase of relative weight of the liver in females were observed. From these results, the main adverse effects of the test substance were estimated to be a hepatocellular injury and an irritant property to gastric mucosa.

The authors of the study concluded that the No-Observed-Adverse-Effect Level (NOAEL) of the test substance under the conditions tested was 21 mg/kg/day since the following adverse effects were observed in the 85 mg/kg groups; decreased spontaneous locomotion in males and females, incomplete eyelid opening and statistically significant decreases of total protein and albumin in males and focal necrosis of fundic mucosa in the glandular stomach in females.

However, the registrant considers the effects observed at 85 mg/kg not to be adverse. The decreased spontaneous locomotion seen at the clinical observations in 4/5 male rats and, on a few occasions only, in 1/5 female rats of the 85 mg/kg group is likely to be secondary to the irritant effect of the substance on the gastrointestinal tract following the oral administration rather than a reflection of systemic toxicity. This is supported by the absence of an effect on spontaneous motor activity during the weekly detailed clinical observations (arena observations) and during the quantitative motor activity assessment conducted at the end of the 28-day treatment period. Haematological parameters are not affected at 85 mg/kg bw. Biochemical parameters: Protein is slightly decreased in males only at 85 mg/kg bw but < 10%. In females there is a slight increase in gamma-GT and in total cholesterol of which the latter decreases again at 340 mg/kg bw. The liver parameters are only slightly affected and can still be considered an adaptive response. An enlarged liver is seen in one male at this dose, which is considered an adaptive change. In females a microscopic effect is seen in the glandular stomach (focal minimal necrosis of the fundic mucosa) in one animal. This effect is considered to be due to the irritant properties of the substance and therefore does not need to be considered for the systemic NOAEL. In summary, the registrant estimates the systemic NOAEL to be 85 mg/kg bw/day.

 

Report on assessing repeated dose toxicity and developmental toxicity of delta-Damascone using alpha-Isomethylionone

Delta-Damascone is an ionone, containing an unsaturated hexylring (hexenyl ring) to which an alkenyl chain with an alpha-beta unsaturated and conjugated ketone is attached. In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i. e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the repeated dose and developmental toxicity of delta-Damascone the analogue approach is selected because for a closely related analogue 90-day repeated dose and developmental toxicity information is available, which can be used for read across to delta-Damascone.

Hypothesis for the analogue approach

Delta-Damascone has similar systemic repeated dose and developmental toxicity as alpha-Isomethylionone based on the similarity in chemical structure, physical chemical properties, reactivity, receptor binding and metabolism between these two substances (see Table 1). Available information: For the target substance delta-Damascone an acute oral toxicity test is available and a 28-day repeated dose toxicity study similar to OECD TG 401 and OECD TG 407, respectively. For the source substance acute toxicity (OECD TG 401), 90-day repeated dose (OECD TG 408) and developmental toxicity information (OECD TG 414) is available.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemicals are shown in the data matrix , including physico-chemical properties and toxicological information, thought relevant for the endpoints that are assessed.

Purity / Impurities

The main impurities are n-alpha-Isomethylionone (CAS no 7779-30-8) (20.9%) and n-betamethylionone (CAS no 127-43-5) (7.9%). Because of the structural similarities of these impurities, it is expected that these impurities do not affect the physico-chemical parameters and human toxicological properties. It is therefore concluded that the impurities will not hinder the read-across (see table 1).

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. It can also be used when the analogues used will have the same or similar metabolites. 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. This application and documentation is presented here.

Structure similarity between delta-Damascone and alpha-Isomethylionone: Delta-Damascone has significant structural homology with alpha-Isomethylionone, both having a cyclohexenyl ring with three methyl groups and a side chain, with a ketone moiety, attached to this ring. The alkenyl side chain of delta-Damascone is a 2-buten-1-one group and of alpha-Isomethylionone a 3-buten-2-one group. Both are alpha-beta unsaturated ketones. Delta-Damascone may exhibit some higher reactivity, on structural grounds, because it does not have the methyl group attached to this alpha-beta unsaturated ketone, which alpha-Isomethylionone has. This methyl group is expected to somewhat hinder the reactivity of the conjugated bond. Another difference between these two chemicals is the switched positions of the alkenyl double bond and the ketone (see table 1).

Toxicokinetic aspects: The physico-chemical properties of delta-Damascone and alpha-Isomethylionone are similar and are therefore expected to have similar absorption and other kinetic behaviour (see data matrix). Both substances have similar water solubilities and vapour pressures, which are relatively low (Table 1). The substances have Log Kow of 4.2 and 4.7, respectively, which illustrates that they are both highly lipophilic.

Metabolism: The metabolic pathway is also expected to be similar (Belsito et al., 2007). The vinyl substituted functional group is expected to be metabolized through reduction to the butenone group to a secondary alcohol, oxidation of the angular methyl groups (becoming alcohols), reduction of the double bond in the exocyclic alkenyl side chain to form dihydro-derivatives and conjugation with glutathione (see metabolic prediction on general ionones, roughly drafted after Belsito et al., 2007). The results of the rat liver S9 simulator of the OECD Toolbox are presented in Appendix 1.

Toxicity aspects: Delta-Damascone and alpha-Isomethylionone have similar toxicity profiles though delta-Damascone may be slightly more toxic for several endpoints as is summarized below.

Acute oral toxicity: Delta-Damascone has a slightly higher acute toxicity (lower LD50) compared to alpha-Isomethylionone and will be classified for acute oral toxicity (see Table 1).

Skin irritation: Delta-Damascone shows skin irritation properties in an in vitro test and need to be classified as such. Alpha-Isomethylionone is considered slightly irritating in an in vivo test, which does not warrant classification and labelling.

Skin sensitization: Delta-Damascone is a strong sensitizer, while the information on alpha-Isomethylionone presents weak sensitization. This difference can be explained by the presence of the methyl group in the alkyl chain in the latter substance, decreasing the reactivity.

Repeated dose toxicity: For delta-Damascone a 28-day study has become available in 2013. In this study the target organ is the liver and a systemic NOAEL of 85 mg/kg bw is derived, though at the highest dose of 340 mg/kg bw also than liver parameters were affected. For alpha-Isomethylionone also the liver is the target organ in the 90-day repeated dose toxicity study. At the highest dose of 500 mg/kg liver effects are seen. At this high dose also an effect on the bone-marrow is seen. At the mid dose of 30 mg/kg bw no effects are observed.

Reproductive toxicity, Fertility: No effects have been seen on fertility (based on the absence of toxicity to the reproductive organs) in the repeated dose toxicity studies of delta-Damascone and alpha-Isomethylionone. Therefore no adverse effects are expected.

Reproductive toxicity, Developmental toxic effects: Alpha-Isomethylionone can be used for read across for this endpoint similarly to what has been presented at repeated dose toxicity. In addition, there are limited indications for delta-Damascone and alpha-Isomethylionone belonging to a group of developmental toxicants as presented in Wu et al. (2013). The only indicator for developmental toxicity one may see is some similarity between the ionones and Vit A, (retinol) of which the chemical structure is presented below. It can be seen that the hexenyl ring of alpha-Isomethylionone shows some similarity with Vit A, though the double bond in the ring is not conjugated with the bonds in the alkenyl side chain, which diminishes the similarity, resulting in < C3 conjugated bonds and is thus not similar to Vit. A (Wu et al., 2013). The 2-position of the ketone group in the side chain is also dissimilar to Vit A. No developmental effects were seen at high doses for alpha-Isomethylionone at doses up to 30 mg/kg bw for which clear effects are seen for Vit A (Hendrikx et al., 2000): doses >= 6 mg/kg bw in monkeys. Delta-Damascone is even more dissimilar to Vit A because of the position of the double bond in this hexenyl ring and the absence of the methyl group attached to this double bond in the ring. Delta -Damascone also has a short alkenyl chain and not a methyl group attached to the alpha, beta-conjugated bond, which indicates even a lower similarity with Vit A.

Uncertainty: Delta-Damascone and alpha-Isomethylionone are similar with regard to structure, physical chemical properties, reactivity, receptor binding, metabolism and toxicity profile. The main difference is the methyl group attached to the alpha-beta conjugated bond in alpha-Isomethylionone, which is expected to diminish its reactivity. This can indeed be seen in the skin irritation and skin sensitization potential being lower. The absence of effects in the gastrointestinal tract of rats treated orally with alpha-Isomethylionone, which are present in rats treated orally with delta-Damascone, may also be an indicator for slightly lower reactivity. In the 28-day repeated dose study with delta-Damascone at 340 mg/kg bw several toxicological effects are seen. These effects other than liver parameters were not seen in the repeated dose toxicity study of the source substance. No effects were seen in the bone-marrow of delta-Damascone treated animals, while some indications were seen during testing with alpha-Isomethylionone, which may be due to the shorter exposure period of the target. For repeated dose an additional assessment factor can be used to account for this expected slightly higher toxicity of delta-Damascone. It has been shown that alpha-Isomethylionone and delta-Damascone are sufficiently similar to use the information for read across.

In addition, the review paper from Belsito et al. (2007) shows that the whole group of ionones and rose ketones have similar toxicological characteristics, indicating that the functional groups are more important for the repeated dose toxicity than their structural dissimilarities. Therefore, the data gaps for delta-Damascone on sub-chronic toxicity and developmental toxicity could be filled in by using the data of alpha-Isomethylionone.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix in Table 1.

Conclusions per endpoint for C&L, PBT/vPvB and dose descriptor

When using read across the result derived should be applicable for C&L and/or risk assessment, cover an exposure period duration comparable or longer than the corresponding method and be presented with adequate and reliable documentation as is presented in the present document. Alpha-Isomethylionone is considered an appropriate read-across candidate to be used to fill in the data gaps on delta-Damascone, which are 90-d repeated dose toxicity and developmental toxicity. The NOAELs for alpha-Isomethylionone for 90-d repeated dose toxicity and developmental toxicity were 30 and >30 mg/kg bw/day, respectively.

Classification and Labelling

Repeated dose toxicity: The effects and NOAEL (85 mg/kg bw) in 28-day repeated dose toxicity study with delta-Damascone do not indicate severe effects and therefore classification and labelling is not needed for this endpoint. Also, the 90-day repeated dose toxicity of alpha-Isomethylionone does not indicate severe repeated dose effects. Though alpha-Isomethylionone has a NOAEL of 30 mg/kg, the effects seen at 500 mg/kg bw (LOAEL) are not considered sufficiently severe to warrant classification and labelling for this endpoint. Therefore delta-Damascone does not need to be classified for 28-day and 90-day repeated dose toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Developmental toxicity: The developmental toxicity is based on alpha-Isomethylionone. Since alpha-Isomethylionone need not to be classified and labelled for this endpoint, delta-Damascone is not considered a developmental toxicant in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

DNEL-derivation: For the DNEL-derivation of delta-Damascone, the NOAELs of 30 mg/kg bw/day for 90-d repeated dose toxicity and developmental toxicity will be used as a starting point. For derivation of the DNEL an assessment factor will be included for uncertainty, because delta-Damascone is considered to be slightly more reactive (for details see DNEL derivation).

Table 1: Data Matrix, Analogue Approach for delta-Damascone with alpha-Isomethylionone

Chemical name	delta-Damascone (Target chemical)	Alpha-Isomethylionone (Source chemical)
Structure
CAS no.	57378-68-4	127-51-5
Molecular Weight	192.3	206.3 1)
Purity	95-100%
PHYSICO-CHEMICAL DATA
Physical state at 20 °C and 101.3 kPa	Colorless to pale yellow liquid	Colorless liquid
Melting Point (°C)	10 (33 calculated1) )	45 (calculated) 1)
Boiling point (°C)	Decomposition before boiling	266.2 1)
Partition coefficient (Log Kow)	4.2	4.6 1)
Vapour pressure (Pa) at 20°C	2.7 (IFF internal data) 3.6 (calculated, EpiSuite)	1.2 (IFF internal data) 1.5 (calculated) 1)
Water solubility (mg/L) at 20°C, pH 7.1	77.2 (13.3 calculated) 1)	16 1)
MAMMALIAN TOXICITY
Acute oral toxicity (LD50)	1821 g/kg bw (mice, Moron, 1980))	8714 LD50 mouse (Hoffmann-Larouch, 1967, RIFM database) > 5 g/kg bw (rat) 1)
Skin irritation	Positive (in vitro test, B.36)	Negative 3)
Sensitisation	Positive EC3 = 0.886%	Positive EC3 = 21.8%
28-day repeated dose toxicity (NOAEL)	85 mg/kg bw
90-d repeated dose toxicity (NOAEL)	Read-across	30 mg/kg bw/day 4)
Developmental toxicity test (NOAEL)	Read-across	> 30 mg/kg bw/day 4)

1) http://www.epa.gov/hpv/pubs/summaries/ionederv/c13660tp.pdf

2) http://www.chemicalbook.com/ProductChemicalPropertiesCB5183650_EN.htm

3) RIFM database, performed according to Annex V of EEC Directive 79/831 (OECD TG 414)

4) Belsito et al., 2007

Belsito, D., Bickers, D., Bruze, M., Calow, P., Greim, H., Hanifin, J.M., Rogers, A.E., Saurat, J.H., Sipes, I.G., Tagami, H., 2007, A toxicologic and dermatologic assessment of ionones when used as fragrance ingredients,  Food Chem Toxicol. 2007;45 Suppl 1:S130-67.

Hendrikxs, A.G., Peterson, P., and Hummler, H., 2000, H., Vitamin A teratogenicity and risk assessment in the macaque retinoid model, Reprod. Toxicol., 14, 311-323.

Wu, S., Fisher, J., Naciff, J., Laufersweiler, M., Lester, C., Daston, G. and Blackburn, K., 2013, Framework for Identifying Chemicals with Structural Features Associated with the Potential to Act as Developmental or Reproductive Toxicants, Chem. Res. Toxicol.,26, 1840-61

 

Appendix 1 OECD Toolbox (3.1.0.21) rat liver metabolism simulator.

Justification for classification or non-classification

According to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, a classification of the substance into Category 2 STOT needs to be considered if a NOAEL obtained in a 90-day oral study with rats is below the classification limit of 100 mg/kg bw/day or a NOAEL obtained in a 28 -day oral study in rats is below 300 mg/kg bw. Although the established NOAELs for both Delta-damascone and Alpha-iso-methylionone are considerably below this limit value, the Regulation however states that classification is not warranted when observed effects are limited to changes in organ weights with no evidence of organ dysfunction and/or clinical observations or small changes in body weight gain, food consumption or water intake that have toxicological importance but that do not, by themselves, indicate ‘significant’ toxicity. This is the case for both tests. In addition, observed effects have been noted only at dose levels which are higher than the classification limits. Therefore classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 is also considered to be not warranted.