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Description of key information

Hexalon repeated dose toxicity is derived from the analogue Galbascone for which a dietary OECD TG 408/422 was performed: NOAEL: 42 mg/kg bw/day (700 ppm)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from an analogue which was tested using an > 90 day OECD TG 422 study
Justification for type of information:
The read across justification is presented in the Repeated dose Endpoint summary and the accompanying file is also attached there.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
42 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
diet 700 ppm
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
137 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
no
Conclusions:
The derived NOAEL is 42 mg/kg bw.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
42 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
For repeated dose toxicity adequate read across is performed from a close analogue Galbasconen for which a sub-chronic study is available. Therefore the information is sufficiently adequate.
System:
urinary
Organ:
bladder

Additional information

The repeated dose toxicity information for Hexalon is derived from Galbascone a close analogue. First the repeated dose of Galbascone will be summarised below including effects on fertility and developmental toxicity. Thereafter the read across justification is based on repeated dose only. The accompanying files are attached in the study record.

The OECD TG 408/422 with Galbascone used for read across to Hexalon (this summary is repeated in the fertility and developmental toxicity section):

Introduction and method: The substance was administered via the diet to male and female Wistar Han rats at dietary concentrations of 250, 700 and 2000 ppm (10 rats/sex/dose level) according to OECD 422 Guideline (nominal ca: 133, 47 and 17 mg/kg bw). Concurrent controls (10 rats/sex) received basal diet without test item. Males were exposed for 10 weeks prior to mating, during mating, and up to termination (for 91 days). The females were exposed for 10 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 103 - 114 days). Analysis of diet preparations showed that the diets were prepared accurately and homogenously, and were stable during storage at room temperature under normal laboratory light conditions in an open container for at least 8 days and in a freezer (≤-15°C) for at least 3 weeks.

The food and test substance intake of 250, 700 and 2000 ppm resulted in 16, 42 and 137 mg/kg bw, using the lower and thus most conservative values of males and females.

Results:Parental effects: There were no adverse changes in in-life parameters (body weight, food consumption, clinical signs and functional observations) across the dose groups. A lower body weight and food consumption was recorded for females treated at 2000 ppm throughout the post-coitum and lactation periods, and for body weight also during the premating period. Compared to controls, mean body weights were about 10% lower than controls, whereas the differences in food consumption were more marked (approximately 30-40% lower than controls throughout the post-coitum and lactation period). This lower food intake was not considered adverse in nature since it occurred in absence of any adverse changes in body weight or clinical appearance, or treatment-related changes in pup body weights.

Heamatology and clinical biochemistry parameters: No adverse effects were observed.

Organ effects: liver: Relative liver weight was increased in males and females with 13% and macroscopic liver enlargement was seen at the high dose in some males. Microscopically a treatment-related but non-adverse hepatocellular hypertrophy was recorded in the liver of males and females treated at 2000 ppm up to slight degree. In the absence of any other indicators of hepatocellular toxicity, these liver changes were not considered to be adverse.

Kidney: In males at 2000 ppm had kidney weights that were approximately 15% higher than controls and were considered to be related to the microscopic renal changes. Higher kidney weights were also recorded for males at 700 ppm (approximately 13% higher than controls), and for females at 250, 700 and 2000 ppm (approximately 13%, 21% and 16% higher, respectively). There were neither corroborative microscopic changes nor clinical biochemical alterations indicative of renal toxicity. Furthermore, kidney weights remained within the range considered normal for this strain of rats of similar age. Therefore, the higher kidney weights for males at 700 ppm and for females at 250 ppm and higher were not considered adverse in nature.

At 2000 ppm (ca 137 mg/kg bw) an adverse histopathological lesion was recorded in the kidneys of males treated at 2000 ppm. In these males, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted. The hyaline droplet accumulation was considered to represent alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats and not in higher mammals, including man (Ref. 6, see end of section). In this study the increased hyaline droplet accumulation in males treated at 2000 ppm was accompanied by indicators of renal tubular damage in the form of granular casts at minimal degree and increased tubular basophilia up to slight degree. The granular casts and the increase in tubular basophilia are considered adverse for rats.

Microscopic examination in female urinary bladder revealed minimal to slight hypertrophy of the urothelium treated at 2000 ppm (ca 137 mg/kg bw). This test item-related change was considered to be adverse in this 90-day study because it was seen in three out of five females, none in the control and it is an effect which does not typically occur as background finding. It does not seem to be a secondary response to irritation by calculi, lower urinary tract obstruction or in association with renal papillary necrosis as these effects were not observed. Therefore the bladder effects are possibly a primary effect of the test item. The mode of action for this effect is unclear and therefore the effect is used for deriving the NOAEL. There are, however, no indications that the kidney function in females was impaired based on other kidney (related) findings. In addition, there were no related findings seen such as morphological lesions (e.g. inflammation or cell death) and it was not seen in males.

Conclusion

Repeated dose systemic effects: The effects on the bladder in the females were considered adverse and therefore the overall NOAEL is set at 42 mg/kg bw.

Fertility: No fertility effects were observed up to and including 2000 ppm, the highest dose level tested. Based on the absence of adverse effects on fertility parameter, the NOAEL on fertility is >=2000 ppm (>=137 mg/kg bw) were seen.

Developmental toxicity: Developmental toxicity parameters were not affected up and including 2000 ppm (equivalent to 168 mg/kg bw, based on females only).Based on the absence of adverse effects on developmental toxicity parameters, the NOAEL developmental toxicity in this study is at least >=2000 ppm (>=168 mg/kg bw).

Assessing repeated dose toxicity of Hexalon (CAS #79-78-7) using information from Galbascone (CAS #56973-85-4)

Introduction and hypothesis for the analogue approach

Hexalon (CAS #79-78-7) has a trimethyl-cyclohexene backbone with an alkyl chain to it. The alkyl chain has an alpha-beta conjugated ketone bond and has an allyl bond at the end of the chain. For this substance, no information on repeated dose toxicity is available.

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 asin vitrotests, QSARs, grouping and read-across. In this case read across will be used for assessing repeated dose toxicity. Galbascone has a similar molecular structure and therefore test information from Galbascone can be used to predict the repeated dose toxicity of Hexalon.

Hypothesis:Hexalon (target) has similar repeated dose toxicity compared to Galbascone (source) resulting in a similar NOAELs because the structure is similar and therefore the repeated dose effects are expected to be similar.

Available information:For Galbascone a dietary sub-chronic study (> 90 days), equivalent to OECD 422 (in compliance with GLP) is available and has reliability of 1.

2. Target chemical and source chemical(s)

Chemical structures of the target and the source are shown in the data matrix.

3. Purity / Impurities

Hexalon contains one main constituent present at ca. 78%, a constituent present at ca. 10% and three additional constituents all present below 5%. Except one (1.3%) all minor constituents of Hexalon have similar structures: a ring with an alkyl chain with a ketone group. This minor one has more a ring type of structure instead of a chain which is not expected to have a more severe repeated dose toxicity compared to the other constituents. Galbascone contains two constituents that are isomers. As a result it is not expected that the impurities of the source and target chemicals affect the read-across justification.

4. 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 “common breakdown products via physical and biological processes, which result in structurally similar chemicals”. 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 read across will be based on a common structural backbone, similar functional groups and the formation of similar breakdown products.

Galbascone is selected as an analogue for Hexalon because this is the most similar substance within the ionone family (Belsito et al. 2007, 2013), for which adequate repeated dose toxicity information is available. The ECHA guidance (2017, RAAF) is considered when appropriate.

Structural similarities and differences:Hexalon has a trimethyl-cyclohexene backbone with an alkyl chain attached to it. The alkyl chain has an alpha-beta conjugated ketone bond and has an allyl bond at the end of the chain. Structural similarities between Hexalon and Galbascone are the alkyl chain attached to the ring, having an allyl group at the end of this chain, and four C-atoms between this allyl group and the alpha-beta conjugated bond. Hexalon has the double bond in conjunction with the ketone group outside the ring while Galbascone has this bond inside the ring.

Toxico-kinetic similarities and differences:Absorption:Hexalon and Galbascone have similar molecular weight and physico-chemical properties indicating similar absorption characteristics (molecular weight (232 and 192 g/mol), log Kow (5.5 and 4.5), and vapour pressure (0.08 and 1.14 Pa at ca. 25 °C), respectively). This indicates that Hexalon and Galbascone will be absorbed similarly via all routes. Hexalon has a higher log Kow compared to Galbascone but this is still in the range of good oral absorption. Hexalon is expected to have a somewhat lower dermal absorption because with a log Kow of 5.5 it will not easily pass the aqueous layers of the skin. Based on the higher molecular weight and the higher log Kow it can be expected that the results found for Galbascone are expected to be conservative for Hexalon.Metabolism:The theoretical metabolic pathways are similar for both substances (Belsito et al.2007 and Belsito et al.2013). Possible primary metabolism may include reduction of the ketone group to a secondary alcohol, hydroxylation/oxygenation of the cyclohexene ring, oxidation of the angular methyl groups, reduction of the double bonds in the exocyclic alkenyl side chain or cyclic portion of the molecule to form dihydro derivatives, and epoxidation of isolated (non-conjugated) double bounds of exocyclic alkyl chains and subsequent reaction with epoxide hydrolases or glutathione transferase. Phase II metabolism for both substances includes conjugation of the hydroxylated metabolites, and conjugation of epoxide metabolites.Excretion:The moderate water solubility of the test substances would limit distribution in the body via the water channels. The log Kow would suggest that the substance would pass through the biological cell membrane. A combination of the possible metabolic routes will result in polar metabolites, which can be excreted via the urine. Effects are seen in the kidney and bladder in the repeated dose study with Galbascone, this confirms that the metabolites are excreted via urine. Any unabsorbed substance will be excreted via the faeces.

Toxico-dynamic aspect such as reactivity:Hexalon and Galbascone possess some Michael addition reactivity (OECD Toolbox) based on their functional alpha-beta conjugated ketone group. The alpha-beta conjugation in the ring of Galbascone is expected to be similar reactive as which has this functional group outside the ring. This is because Hexalon has the double bond is only one methyl group away from the ring and the ring actually may somewhat sterically hinder the reactivity at the double bond position when Michael addition reactivity is considered. Both Hexalon and Galbascone have a double bond at the end of the alkyl chain and the formation of a metabolite at this spot, will not present difference in reactivity. The additional double bond in the ring of Hexalon is not very reactive though it may auto-oxidise and form a hydroperoxide.

Experimental datasimilarity and difference:The acute and local endpoints indicate a similar reactivity and no other information is available for comparison.

Uncertainty of the prediction:There is no remaining uncertainty, in view of similarities in structure, toxico-kinetic (absorption and metabolism) and anticipated toxico-dynamic profile (reactivity) the read across is justified.

5. Data matrix

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

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

Repeated dose results were obtained using read across from Galbascone for which asub-chronic study (> 90-days) including screening for reproductive toxicity study (OECD 422) was performed. The NOAELs for repeated dose toxicity was determined to be 42.0 mg/kg bw/day based on slight effects in the bladder, for which classification and labelling is not needed. In view of the presented analogy this information can be used for Hexalon.

Final conclusion on hazard, C&L, DNEL and risk characterization

For Hexalon a NOAEL of 42 mg/kg bw is derived for which no classification and labelling is needed according the EU CLP (1272/2008 and its updates). A conversion of the NOAEL is not needed because Galbascone has the lower molecular weight and therefore the NOAEL can be conservatively used for Hexalon. 

Data matrix for to assess the repeated dose toxicity of Hexalon using read across from Galbascone

Common names

Hexalon

Galbascone

Chemical structures*

Chemical name

1-(2,6,6-Trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one

1-(5,5-dimethyl-1-cyclohexen-1-yl)pent-4-en-1-one

REACH registration

2018

2018

EINECS

201-225-9

260-486-7

CAS no

79-78-7

56973-85-4

Smiles

O=C(C=CC(C(=CCC1)C)C1(C)C)CCC=C

CC1(C)CC(=CCC1)C(=O)CCC=C

Molecular weight (g/mol)

232

192

Appearance

Liquid

Liquid

Physico-chemical data

 

 

Melting point, °C

-20

-20

Vapour pressure, Pa

0.08

1.14

Water solubility, mg/l

79

89

Log Kow

5.5

4.5

Human health

 

 

Acute oral tox (mg/kg bw)

LD50 = 9500 (similar to OECD 401)

LD50 > 2000 (equivalent to OECD 401)

Acute dermal tox (mg/kg bw)

LD50 > 5000 (similar to OECD 402)

LD50 > 2000 (equivalent to OECD 402)

Repeated dose toxicity (mg/kg bw/day)

Read across

NOAEL = 42.0 (OECD 422)

*Chemical structures are presented in the read across document attached

 

References:

D. Belsito, D. Bickers, M. Bruze, P. Calow, H. Greim, J.M. Hanifin , A.E. Rogers, J.H. Saurat, I.G. Sipes, H. Tagami., 2007. toxicologic and dermatologic assessment of ionones when used as fragrance ingredientsFood and Chemical Toxicology 45 (2007) S130–S167.

 

Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M.L., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013, A toxicological and dermatological assessment of alkyl cyclic ketones when used as fragrance ingredients, Food and Chemical Toxicology 62, S1-S44.

 

Justification for classification or non-classification

The NOAEL of the substance is 700 ppm (corresponding with 42 mg/kg bw/day) and the LOAEL of 2000 ppm (equivalent to 137 mg/kg bw), of which the latter is above the classification and labelling limits. In addition, the effects seen (hypertrophy of the urothelium of the urinary bladder) in females are insufficiently severe for classification and labelling. Therefore, classification of the substance is not warranted according to EU CLP (1272/2008 and its updates).

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