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Administrative data

Description of key information

No data on repeated dose toxicity of delta-damascone are available. However, based on the reliable 90-day oral study with its structural analogue alpha-iso-methylionone (EC name: 3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one), a NOAEL of 30 mg/kg bw/day is established and will be used for DNEL derivation, based on follicular cell hypertrophy in the thyroid, increases in absolute and relative kidney and spleen weight and an increased creatinine level at the next dose level. Route-to-route extrapolation will be used for DNEL derivation for dermal and inhalation routes of exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
30 mg/kg bw/day

Additional information

Repeated dose, oral:

A reliable 90-day study, performed according to OECD Guideline 408 and under GLP, was available with the appropriate read-across candidate alpha-iso-methylionone (EC name:3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one). The substance was administered by oral gavage at dose levels of 500, 30 and 5 mg/kg bw/day for a period of ninety consecutive days to groups of 10 male and female rats. After this period, animals were sacrificed and the gross pathological, histopathological, haematological and clinical chemistry examinations were performed.

Clinical signs and mortalities

There were no mortalities or adverse clinical signs noted. Animals of either sex treated with 500 mg/kg bw/day did show instances of noisy respiration and transient increased salivation together with hunched posture and tiptoe gait. Such changes are commonly observed following oral administration of an unpalatable or slightly irritant test material formulation and are therefore considered not to be indicative of systemic toxicity.

Clinical chemistry

Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol compared to control animals, as well as a statistically significant reduction in aspartate aminotransferase. Males from this treatment group also showed a statistically significant increase in plasma albumin and a statistically significant reduction in plasma chloride concentration, whilst females showed a reduction in alkaline phosphatase. Whilst some of these parameters are an indication ofdisturbed liver function, a true elevation in albumin is unlikely in the absence of any evidence of dehydration, because albumin synthesis normally occurs at close to the maximum possible rate. Furthermore, the elevation in cholesterol was not supported by any other evidence of obstructive liver damage. An increase in creatinine may suggest an effect on kidney function. However, there was no concomitant change in electrolyte balance but, in view of the significant changes seen in 500 mg/kg bw/day kidney weights, a toxicologically significant effect cannot be entirely ruled out. Females from all treatment groups showed a statistically significant reduction in plasma bilirubin. However, the majority of individual values were within the normal range for rats of the strain and age used and in the absence of a dose related response the intergroup differences were considered of no toxicological importance.

Haematology

There were no treatment-related changes in the haematological parameters measured. Statistical analysis revealed no significant intergroup differences.

Organ weights

Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in absolute and relative liver and kidney weight. The statistically significant increase in relative liver weight was also observed in females treated with 30 mg/kg bw/day. Histopathologically, 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, while no changes were noted in females treated with 30 mg/kg bw/day. 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. Animals of either sex treated with 30 mg/kg bw/day showed a statistically significant reduction in absolute and relative heart weight. All individual values were however within the normal range for rats of the strain and age used and in the absence of a dose related response the intergroup differences were considered to be of no toxicological importance. Males treated with 5 mg/kg/day showed a statistically significant increase in absolute and relative adrenal weight. In the absence of a dose-related response or any histological correlates these intergroup differences were considered not to be toxicologically significant.

Males treated with 500 mg/kg bw/day also showed statistically significant increases in spleen weight both absolute and relative to terminal bodyweight. However, this effect was not accompanied by any histopathological changes, minimizing its toxicological significance. However, microscopic examinations of bone marrow did reveal a higher incidence of lower grades of severity of adipose infiltration in these animals, which may suggest a subtle effect on haemopoeisis. The importance of these findings is however lessened by the absence of supporting changes in the haematological parameters.

Histopathology

Liver: 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 bw/day (p< 0.05 for males; p< 0.001 for females). Increased hepatic activity of this type and in the absence of associated inflammatory or degenerative changes is considered a normal adaptive biological response to xenobiotics and is usually the result of detoxification mechanisms involving hepatic enzyme induction. Organ weight data supported this finding with increased absolute and relative liver weights detected in this treatment group.

Kidneys: A greater incidence of higher severity grades of globular accumulations of eosinophilic material was observed in the tubular epithelium of 3/10 (minimal), 5/10 (slight) and 1/10 (moderate) males treated with 500 mg/kg bw/day (p< 0.01) or in 5/10 (minimal) and 4/10 (slight) males treated with 30 mg/kg bw/day (p< 0.05), but not at 5 mg/kg/day. Such accumulation is a well documented effect, peculiar to male rats, 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 bw/day and one male treated with 30 mg/kg bw/day also exhibited associated higher grades of tubular basophilia.

Thyroid: A higher incidence of follicular cell hypertrophy was seen in relation to treatment for 7/10 (minimal) males treated with 500 mg/kg bw/day (p< 0.01). Thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. It is conceivable that conjugating hepatic enzymes may have been induced as a response to the test material, therefore increasing thyroxine excretion and stimulating compensatory thyroxine stimulating hormone and thyroxine production, possibly resulting in the microscopic changes identified.

Bone marrow: 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 3/10 (slight) males treated with 500 mg/kg bw/day (p< 0.01).

Conclusion

Based on these results, the NOAEL for systemic toxicity has been set at 30 mg/kg bw/day, based on increases in absolute and relative kidney and spleen weight, increased creatinine levels and follicular cell hypertrophy in the thyroid at the next dose level. This NOAEL will be used as a point of departure for DNEL derivation for delta-damascone. In order to account for possible uncertainties due to the use of read-across an additional safety factor of 3 will be applied for DNEL derivation for delta-damascone.

Repeated dose; dermal

In Column 1 of REACH Annex IX it is stated that a “sub-chronic toxicity study (90-day), one species, rodent, male and female, most appropriate route of administration, having regard to the likely route of human exposure” needs to be conducted. Because of the relatively low vapour pressure of the liquid, the dermal route is considered the most likely route of human exposure. Reliable data on 90 -day repeated dose toxicity via the oral route are available and data on 90-day repeated dose toxicity via the dermal route with a reliability KC=3 are available. Based on the available toxicokinetic and metabolism data, it is concluded that route-to-route extrapolation from the oral to the dermal and inhalation route is appropriate. Therefore, 90-day repeated dose testing via the dermal route is not needed.

Repeated dose; inhalation

In Column 1 of REACH Annex IX it is stated that a “sub-chronic toxicity study (90-day), one species, rodent, male and female, most appropriate route of administration, having regard to the likely route of human exposure” needs to be conducted. Because of the low vapour pressure of delta-damascone, exposure via the inhalation route is considered of less relevance. As such, the dermal exposure is considered the most appropriate route of administration, having regard to the likely route of human exposure. Data on 90-day repeated dose toxicity via the oral route are available and based on the available toxicokinetic and metabolism data, it is concluded that route-to-route extrapolation from the oral to the inhalation route is appropriate. Therefore, 90-day repeated dose testing via the inhalation route is not needed.

 


Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: spleen; glandular: thyroids; urogenital: kidneys

Justification for classification or non-classification

Although the NOAEL of 30 mg/kg bw/day, obtained in a 90-day study with rats with the structural analogue of delta-damascone, alpha-iso-methylionone, is marginally below the cut-off value of 50 mg/kg bw/day established by EU Directive 67/548/EEC for classification as harmful: risk of serious damage to health by prolonged exposure (R48/22), the classification is considered to be not warranted, as the increase in absolute and relative spleen weight for males was not accompanied by any pathological changes and the haemopoeisis effects were not accompanied by changes in haematological parameters.

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. Although the established NOAEL is 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 alpha-iso-methylionone. In addition, observed effects have been noted only at a dose level which is 16 times higher than the established NOAEL. 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.