Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate

Endpoint summary

Description of key information

The NOAEL of Applelide, being a structural analogue of CP Formate, is considered to be 1000 mg/kg in an OECD TG 407 under GLP, 28-day repeated dose toxicity study in rats, which is considered the key study. The NOAEL of CP Butyrate, also a structural analogue of CP Formate, is considered to be 3000 mg/kg in a 28-day repeated dose toxicity study in rats (similar to OECD TG 407). This latter is the supporting study because of less parameters being investigated.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant study, followed OECD guidelines, well conducted with no deviations.

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

An OECD 422 study with CP Formate will start soon. It is expected that the final report will be available in Q2 2023. When the data comes available, the studies with the structural analogues Applelide and CP Butyrate will be removed and replaced with the OECD 422 study with CP Formate. In addition, the human exposure assessment will be adjusted accordingly.

 

In summary, the available information indicates no repeated dose toxicity at 1000 mg/kg bw, being the limit dose for this endpoint.

No studies on the repeated dose toxicity of CP Formate were available. 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. Two repeated dose toxicity tests were available for two analogues: Applelide and CP Butyrate of which Applelide will be used as key study. First the executive summary of the key and supporting studies are presented and thereafter the read across justification.

Key: 28 -day repeated dose toxicity study on Applelide

In a study performed according to OECD TG 407 and in compliance with GLP, Applelide was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no treatment-related deaths during the study. No treatment related changes in functional performance parameters, sensory reactivity, bodyweight gain, food consumption, water consumption, haematological parameters, blood chemical parameters, and urinalysis parameters were observed. Animals treated with 1000 mg/kg/day developed transient increased salivation around the time of dosing and approximately one hour after dosing and instances of noisy respiration and stained fur or fur loss. Hunched posture was seen in 1000 mg/kg/day animals from Day 21 and episodes of tiptoe gait were observed in two females treated with 1000 mg/kg/day on Day 26 only. All findings regressed in recovery 1000 mg/kg/day animals. An increase in liver weight was detected in animals treated with 1000 mg/kg/day. Four males treated with 1000 mg/kg/day showed pale kidneys. Centrilobular hepatocyte enlargement was observed in relation to treatment for rats at 1000 mg/kg/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive. Globular accumulations of eosinophilic material, with associated renal tubular basophilia in some instances, were observed in the tubular epithelium male rats dosed at 1000 mg/kg/day, 150 mg/kg/day, or at 15 mg/kg/day. There was partial regression of the condition among recovery 1000 mg/kg/day animals after completion of the fourteen day recovery period. 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. A higher incidence of follicular cell hypertrophy was seen in relation to treatment for female rats dosed at 1000 mg/kg/day, but not at any other dose level. The condition was observed to have regressed among Recovery 1000 mg/kg/day animals following an additional fourteen days without treatment.

In conclusion, oral administration of the test material, to rats, by gavage, for a period of twenty-eight consecutive days resulted in treatment-related but non-adverse changes in either sex at a dose level of 1000 mg/kg/day and in males treated with 150 or 15 mg/kg/day. There was no effect of treatment in females treated with 150 or 15 mg/kg/day. The treatment-related renal changes seen in male rats treated with 150 or 15 mg/kg/day were consistent with a well-documented condition known as hydrocarbon nephropathy, which only occurs in male rats and is not indicative of a hazard to human health. Furthermore, the effects seen in animals treated with 1000 mg/kg/day were confined to minimal clinical observations and adaptive, reversible liver and thyroid changes so the NOAEL was considered to be 1000 mg/kg/day.

Supporting: 28 -day repeated dose toxicity study on CP Butyrate

In a 4 week oral toxicity study in CD rats similar to OECD TG 407, CP Butyrate was administered by gavage at dosage levels of 100, 300, 1000 and 3000 mg/kg bw/day. A control group was similarly treated with the corn oil vehicle. Five male and 5 female rats were used at each dosage level. The rats were observed twice daily for overt signs of toxicity and for mortality. Detailed observations were recorded weekly. Individual body weights and food consumption were recorded weekly.

Excessive salivation was noted for a few rats at the 1000 mg/kg bw/day dosage level and for all but one of the rats at the 3000 mg/kg bw/day dosage level. Male rats at the 3000 mg/kg bw/day dosage level showed very slightly lower gains in body weight (-6%) compared to control male rats. Food consumption values were similar for control and treated rats. No gross lesions which were considered compound related were seen in rats which sacrificed at termination. No evidence of toxicity attributable to treatment was observed at the highest dose of 3000 mg/kg bw/day.

In conclusion, the NOAEL in the 28-day repeated dose toxicty study is considered to be 3000 mg/kg bw/day.

 

Besides the above information also the repeated dose toxicity information of CP Butyrate during the developmental toxicity (similar to OECD TG 414) can be included. At the highest dose of 3000 mg/kg bw effects on body weight was seen in the dams. The systemic NOAEL of 1000 mg/kg bw is derived and is discussed at the developmental toxicity section.

 

Reporting on the read-across to CP Formate from Applelide and CP Butyrate for repeated dose toxicity.

Introduction and hypothesis for the analogue approach for repeated dose toxicity

CP Formate is a formate ester of 1,3,3,-methyl cyclohexanol. For this substance no repeated dose toxicity data are 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 as in vitro tests, QSARs, grouping and read-across. Therefore, test information from two analogues will be used to draw a conclusion on repeated dose toxicity for CP Formate.

Hypothesis: CP Formate is expected to have similar repeated dose toxicity considering target organs and NOAEL compared to its analogues, because these substances are similar in structure and will metabolise into similar metabolites after systemic exposure.

Available information: For the analogue Applelide a reliable 28-day repeated dose toxicity test in rats is available according to OECD TG 407. For CP Butyrate also a 28-day study is available but this study receives a reliability of 2, because less parameters have been considered in comparison with the present guidelines.

Target and Source chemical(s)

The target and the source chemicals are presented in the data matrix below including their chemical structures, CAS numbers and physico-chemical properties. The source chemicals are Applelide and CP Butyrate.

Purity / Impurities

CP Formate is a multi-constituent composed of 65 to 85% 1-(3,3-dimethylcyclohexyl) ethyl formate and 8 to 18% 2,6,6-trimethylcycloheptyl formate. The major constituent of CP Formate is a formic-ester of ethyl-substituted cyclohexanol. The minor constituent has a similar functional group but a larger ring structure of 7- C atoms (see IUCLID section 1). This similarity in the functional group will present similar reactivity. The larger ring just contains C-atoms, without any functional groups. Therefore this difference in ring structure in the target will not influence the systemic toxicity. Furthermore, it is not expected that any impurities present in CP Formate will significantly affect the toxicity, because of their similarity in structure and presence < 5%.

Applelide is also a multi-constituent. The main constituent is presented in the data matrix. The backbone of the impurity of Applelide is the same as for the minor constituent of CP Formate and therefore the toxicity of this minor constituent also has been considered during repeated dose testing.

Analogue approach justification

According to section 1.5 of REACH Annex XI read across can be used to replace testing when the similarity can be based on a common functional group and/or a similar metabolite. When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

The read across for CP Formate will be based on similarities in backbone and functional groups of the target and the source chemicals. The target and source chemicals also metabolise into similar metabolites after systemic exposure.

Structural similarities and differences: The backbone of the target and the source consists of a cyclohexyl ring with an ethyl group at which an ester group is attached on the second C of the ethyl-group. The source chemical, Applelide, has two ester groups attached to the ethyl-cyclohexyl ring. These differences in structure and the relation to the toxicological profile of these substances are discussed below. The source chemical CP Butyrate has a C3 alkyl group attached to the ester, while the target, CP Formate, does not have this short alkyl chain. CP Butyrate is missing the two methyl groups at the third position of the ring. These two methyl groups are inactive considering the metabolic behaviour. They are also unreactive and will not influence the repeated dose toxicity.

Toxico-kinetics relevant for repeated dose toxicity: For repeated dose toxicity the absorption, distribution, metabolisation and excretion (ADME) potential of the target and the source chemicals should have similarities to be able to use them for read across. Physico-chemical parameters such as melting point, water solubility and log Kow are the main parameters to assess absorption and distribution properties. It can be seen that for the target and the source chemicals these physico-chemical parameters are sufficiently similar to expect a similar absorption and distribution profile. All substances are liquids, have water solubilities between 10-100 mg/l and log Kow values between 3.5 and 5.

The target and source chemicals will also metabolise similarly. The ester bond will be readily cleaved by carboxylesterases in the gut and liver (and other organs) into their respective alcohols as has been presented by the WHO (2000) for similar esters as CP Formate, by Yamada et al. (2013 for allylic esters) and/or presented in Toxicological handbooks. CP Formate and Applelide metabolise into 1,3,3-dimethylcyclohexanol (CAS no 25225-09-6). For CP Butyrate the respective alcohol is 1-cyclohexyl ethanol (CAS no: 1193-81-3). The ester cleavage is expected to occur at the same rate, because the H-atom group of CP Formate will not hinder this cleavage. The double ester cleavage of the Applelide is expected to be similarly fast because the acetate and propionate are short chains as is the butyl chain of CP Butyrate, because these chains are small and not branched (Yamada et al., 2013 and WHO, 2000). These simple esters with small C-chains and without additional functional groups metabolise following the scheme presented below. The two methyl groups at the ring of CP Formate are non-reactive and will therefore not influence the metabolic profile. The resulting secondary alcohols will be conjugated with glucuronic-acid and then excreted via the urine or the bile (WHO, 2000, Belsito et al., 2008). In addition, the WHO (2011) concluded for a structurally similar substance, 3,3,5-Trimethylcyclohexyl acetate with CAS 67859-96-5, that it would be metabolised into innocuous products. Similar innocuous metabolites are expected for CP Formate.

 

 

Figure 1: The ester cleavage of CP Formate (target), Applelide and CP Butyrate (source chemicals) occurring in the gut and in the liver (and other organs) resulting in the presented alcohols and acids.

Toxicological properties for repeated dose toxicity: Applelide has a low order of repeated dose toxicity. Salivation around the time of dosing and some adaptive liver and thyroid changes were seen in Applelide at the highest dose (1000 mg/kg bw). In addition, alpha-hydrocarbon nephropathy occurred in male rats at all dose levels (15, 150 and 1000 mg/kg bw). This male-rat specific nephropathy is considered not to be indicative of a hazard to human health. CP Butyrate also shows a low order of repeated dose toxicity. The NOAEL was set at 3000 mg/kg bw in the repeated dose toxicity study in rats. Only salivation and a very slightly lower gain in body weight were observed in the 3000 mg/kg bw dosage group. It should be noted that the number of endpoints in this study was limited (haematology, clinical chemistry, organ weight measurements and histopathology were not conducted). During the developmental toxicity study (similar to OECD TG 414) CP Butyrate showed ataxia and reduced body weight gain at 3000 mg/kg bw. This reduced weight gain was also observed in dams treated with 1000 mg/kg bw but was only minimal (< 6 %). The maternal NOAEL was therefore 1000 mg/kg bw in the developmental toxicity study with CP Butyrate. Based on these two-28-day repeated dose toxicity studies and maternal effects in the developmental toxicity study it can be seen that these analogues have a low order of toxicity. Therefore CP Formate also will have low repeated dose toxicity.

No repeated dose toxicity information is available on the respective alcohols (Belsito et al., 2008). The metabolites formic acid (CP Formate), acetic acid (Applelide), propionic acid (Applelide) and butyric acid (CP Butyrate) are all natural constituents of the body and are consumed via food. Therefore the toxicity of these acids does not have to be discussed separately because the metabolisation with carboxylesterases will occur in a controlled way and in a buffered cell medium. Local toxicity e.g. due to acidity will therefore not occur.

It can be concluded that the NOAEL of Applelide of 1000 mg/kg bw/day, which is in line with the value for CP Butyrate, can be used for CP Formate.

Remaining uncertainties: The additional ester group of Applelide, compared to CP Formate, can give some additional reactivity because the C-atom in between the ester will be somewhat electrophilic due to the two ester-bonds at each side. These groups withdraw electrons from this middle C (becoming positively charged) and making this C-atom more susceptible for nucleophilic attack (e.g. electronegative sites in the proteins). Despite this reactivity Applelide is not a skin sensitiser in a LLNA test at 100%. This reactivity may have induced the positive result in the chromosomal aberration assay at the highest dose of 10mMol/ml. This result can be disregarded because the maximum advised doses in this test is currently 1 mMol/ml (Galloway et al., 2011) and the result is unlikely to be relevant during normal exposure. The metabolisation of the double ester of Applelide may have resulted in some adaptive changes in the liver which may be somewhat stronger for Applelide compared to CP Formate and/or CP Butyrate. Therefore the repeated dose effects seen in Applelide are expected to be similar or less for CP Formate.

Data matrix

The chemical information, the physico-chemical and human toxicological properties of CP Formate and its analogues relevant for repeated dose toxicity are presented in the data matrix below.

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

The results presented above show that the analogues of CP Formate have a low order of toxicity. NOAELs of 1000 and 3000 mg/kg bw for Applelide and CP Butyrate, respectively, have been derived in sub-acute oral toxicity studies in rats. For Applelide some adaptive liver changes have been observed as well as alpha-hydrocarbon nephropathy. These effects are also expected for CP Formate. Furthermore, a maternal NOAEL of 1000 mg/kg bw during a developmental toxicity study is derived for CP Butyrate. Based on the results of the repeated dose toxicity tests of Applelide and CP Butyrate a NOAEL of 1000 mg/kg bw can be derived for CP Formate. This means that CP Formate does not need to be classified for repeated dose toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Furthermore, based on the absence of adverse effects at 1000 mg/kg bw the conclusion of “No hazard identified” for repeated dose toxicity will be derived.

Data matrix for CP Formate and analogues for deriving its repeated dose toxicity.

CHEMICAL NAME	l-Cyclo-citronellene formate (CP Formate)	Propanedioic acid, 1-(3,3-dimethylcyclohexyl) ethyl, ethyl ester (Applelide)	Butanoic acid, 1-cyclohexylethyl ester (CP Butyrate)
CAS	25225-08-5	478695-70-4	63449-88-7
Molecular structure#
Empirical formula	C11H20O2	C15H26O4	C12H22O2
Molecular weight	184.3	270.4	198.3
Physico-chemical properties	Liquid	Liquid	Liquid
Vapour pressure at 25˚C (Pa)	13.4	0.1	4.75 (EpiSuite)
Water solubility at 20˚C (mg/L)	26	13.3	6 (EpiSuite)
Partition coefficient log Kow	3.8	4.9	4.5 (EpiSuite)
Human health effects
Acute oral toxicity mg/kg bw	>5000	>2500	>5000
Acute dermal toxicity mg/kg bw	>5000	>2000	No data
Skin irritation	Not an irritant (OECD TG 439)	Not an irritant (OECD TG 404)	No data
Eye irritation	Not an irritant (OECD TG 438)	Not an irritant (OECD TG 405)	No data
Skin sensitisation	Read across Using CP Acetate	Negative up to 100% (OECD TG 429)	Negative (HRIPT up to 100%)
Repeated dose toxicity	Read across	1000 mg/kg bw (OECD TG 407)	3000 mg/kg bw (similar to OECD TG 407)
Maternal repeated dose toxicity			1000 mg/kg bw (similar to OECD TG 414)
Ames test	Negative (OECD TG 471)	Negative (OECD TG 471)	Negative (OECD TG 471)
In vitro cytogenicity	Negative (OECD TG 487)	Negative (OECD TG 473)	No data
In vitro gene-mutations	Negative (OECD TG 476)	No data	No data

#Molecular structures can be seen in the attached document.

References

Belsito, D., Bickers, D., Bruze, M., Calow, P., Greim, H., Hanifin, J.M., Rogers, A.E., Saurat, J.H., Sipes, I.G., Tagami, H., 2008, A toxicologic and dermatologic assessment of cyclic and non-cyclic terpene alcohols when used as fragrance ingredients, Food and Chemical Toxicology, 46, S1-71.

Galloway, S., Lorge E, Aardema M.J., Eastmond, D., Fellows, M., Heflich, R., Kirkland, D., Levy, D.D., Lynch, A.M,, Marzin, D., Morita, T., Schuler, M., Speit, G., 2011, Workshop summary: Top concentration for in vitro mammalian cell genotoxicity assays; and report from working group on toxicity measures and top concentration for in vitro cytogenetics assays (chromosome aberrations and micronucleus), Mutat Res., 723, 77-83, http://www.ncbi.nlm.nih.gov/pubmed/21255675.

WHO, 2000, Evaluation of certain food additives, Technical Report Series 891, page 53/54, http://whqlibdoc.who.int/trs/WHO_TRS_891.pdf. 

WHO, 2011, Safety evaluation of certain food additives, 73rd meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), http://whqlibdoc.who.int/publications/2011/9789241660648_eng.pdf

Yamada, T., Tanaka, Y., Hasegawa, R., Sakuratani, Y., Yamada, J., Kamata, E., Ono, A., Hirose., A., Yamazoe, Y., Mekenyan, O., Hayashi, M., 2013, A category approach to predicting the repeated-dose hepatotoxicity of allyl esters, Reg. Toxicol. Pharmacol, 65, 189-195.

Justification for classification or non-classification

Based on the results of the sub-acute oral toxicity studies in rats with Applelide and CP Butyrate, both structural analogues of CP Formate for repeated dose toxicity, CP Formate does not need to be classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.