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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

CP Formate is expected not to present adverse effects on fertility based on absence of effects on gonads in two 28-day studies of two close analogues, Applelide and CP Butyrate. This is further supported by the absence of concern presented by the WHO (2011) who say that a close structurally related analogue will result in innocuous metabolites.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The information on the two analogues during 28-repeate dose toxicity studies and the WHO 2011 information is sufficiently adequate to cover this endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In accordance with Column 2 of REACH Annex VIII, a screening for reproductive/developmental toxicity study does not have to be performed when a pre-natal developmental toxicity study is available. Furthermore, according to Column 1 of REACH Annex IX, a two-generation study does not need to be conducted when a 28-day study does not indicate adverse effects on reproductive organs or tissues. In the available repeated dose toxicity study with Applelide no adverse effects were observed on the reproductive organs up to the highest dose tested (1000 mg/kg bw): reproductive organ weights were not altered and macroscopically no alterations were observed. Also in the supporting study of CP Butyrate no effects on gonads were seen up to 3000 mg/kg bw.

The read across justification is presented under 'Effects on developmental toxicity'.

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.

Effects on developmental toxicity

Description of key information

Treatment of pregnant rats with the analogue CP Butyrate caused maternal and developmental toxicity at a dosage of 3000 mg/kg bw. The NOAELs were determined to be 1000 mg/kg bw for both maternal and developmental toxicity being the limit dose in the current guidelines.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The selected study is performed with CP Butyrate, a sufficient closely related analogue of CP Formate to cover this endpoint.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

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.

 

No studies on the developmental 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. One developmental toxicity test is available for the analogue CP Butyrate. First these experimental data will be discussed and thereafter the read across justification will be presented.

Developmental/teratology study with CP Butyrate

In a teratology study conducted similar to OECD TG 414, Charles River CD rats were dosed (orally) at levels of 0, 100, 300, 1000 or 3000 mg/kg/day on days 6 through 15 of gestation. Maternal body weights were determined on days 0, 6, 15 and 20 of gestation, and observations of maternal behavior were made daily. Fetuses were delivered by caesarean section on day 20 of gestation. The number of viable fetuses, corpora lutea, implantation sites, still births and resorptions were determined. Fetuses were weighed and examined for external, skeletal and visceral anomalies.

All rats in the 3000 mg/kg dosage group showed signs of ataxia at various times during the treatment period. One rat in this dosage group died after the second treatment, which is unlikely to be related to treatment. Brief signs of ataxia were also observed in two rats of the 1000 mg/kg bw dose group. There was a moderate reduction in mean body weight gain during the treatment period and a slight reduction after the treatment period in the 1000 mg/kg bw/day group. Reduced body weight during the first three days of treatment and reduced mean body weight gains was seen in the 3000 mg/kg bw dosage group. These reduced body weight gains resulted in a decreased body weight. There were no significant differences in the number of nonviable fetuses, corpora lutea, implantations or in the male to female sex ratios. An increase in post implantation loss was noted in the 3000 mg/kg bw group. A significant decrease in mean fetal body weights was also seen in this group. A significant increase in the number of fetuses with bent ribs and with reduced ossification of the skull were seen in the 100 mg/kg dosage group. However, the number of these effects present in higher dosage groups were comparable to controls. No malformations were seen in the 3000 mg/kg dosage group. In conclusion, treatment of pregnant rats with the test substance caused maternal and developmental toxicity at a dosage of 3000 mg/kg bw. The NOAEL was determined to be 1000 mg/kg bw for maternal and developmental toxicity. The highest level tested in the above study was above the current maximum exposure level for a limit test.

 

Reporting on the read-across to CP Formate from Applelide and CP Butyrate for fertility and developmental toxicity, respectively

Introduction and hypothesis for the analogue approach for fertility and developmental toxicity

CP Formate is a formate ester of 1,3,3,-methyl cyclohexanol. For this substance no reproductive and developmental 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 analogues will be used to derive a NOAEL for fertility and for developmental toxicity for CP Formate.

Hypothesis: CP Formate is expected to have similar fertility and developmental toxicity 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, which includes the assessment of the reproductive organs. A similar supporting study is available of CP Butyrate. For CP Butyrate also a developmental toxicity study is available similar to OECD 414, with a reliability of 2.

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 reproductive toxicity. Furthermore, it is not expected that any impurities present in CP Formate will significantly affect this 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.

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 in similar metabolites after systemic exposure. Therefore the information from the analogues can be used to estimate the reproductive toxicity of CP Formate.

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 2^ndC 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 3^rd position of the ring. These two methyl groups are inactive considering the metabolic behaviour and there are no indications these influence the reproductive profile.

Toxico-kinetics relevant for fertility and developmental toxicity: For fertility and developmental 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 carboxyl esterases in the gut and liver 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 respective acids are formic, acetic, propionic and butyric (see scheme below). The ester cleavage is expected to occur at the same rate, because the H-atom group of CP Formate will not hinder this cleavage nor will the straight chain butyl group because these chains are small and not branched. The double ester cleavage of the Applelide is expected to be similarly fast because the acetate and propionate are also short chains. These simple esters, with small C-chains and without additional functional groups will 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).

 

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

 Toxicological properties for fertility and developmental toxicity: Applelide and CP Butyrate did not show any effects on fertility in the 28-day repeated dose toxicity study. CP Butyrate did not show developmental toxicity at 1000 mg/kg bw but pup weight was decreased at 3000 mg/kg bw in the presence of maternal effects. No reproductive 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 reproductive toxicity of these acids does not have to be discussed separately, because the metabolisation with carboxyl esterases will occur in a controlled way and in a buffered cell medium. Local toxicity e.g. due to acidity will therefore not occur.

Based on the absence of fertility effects in the repeated dose toxicity study of the analogue Applelide and CP Butyrate no fertility effects are expected for CP Formate. Based on the absence of developmental effects of the analogue CP Butyrate no developmental effects on CP formate are expected either.

In addition, CP Formate was negative in all three standard in vitro genotoxicity assays further diminishing reproductive and developmental toxicity effects via genotoxicity.

Structure-activity relationship considerations: The type en number structure activity relationships for estimating fertility are limited and are partly related to genotoxicity e.g. epoxides or epoxide former such as benzene (DeVito, 1996). CP Formate and its metabolites do not have such alerts further diminishing the effects on fertility and developmental toxicity.

Other considerations: Blackburn et al (2011) has developed a decision scheme which indicates the (absence of) a concern for reproductive toxicity. Entering the scheme for CP Formate the steps relevant are: CP Formate 1) is an organic compound; 2) contains a cyclic ring but; 3) is not a polycyclic or fused ring and thus CP Formate has: 4) “No known precedent reproductive or developmental toxic potential”.

Remaining uncertainties: The CP Formate information on fertility data may seem somewhat limited because these are only based on an OECD TG 407 study of the analogue Applelide in which reproductive organs are assessed. This information is still considered to be sufficient, because Applelide and CP Formate result in the same alcohol and will be readily excreted due to its water solubility (circa 1300 mg/l, EpiSuite estimation) and due to glucuronation of its alcohol (WHO, 2000), which will further increase the water solubility. 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.

No fertility effects are seen at doses as high as 1000 mg/kg bw, while CP Formate is readily absorbed and overloading the glucuronic pathway may have occurred at these high doses. Therefore no fertility effects are expected during more long-term exposure. For developmental effects CP Butyrate is a reliable analogue with a similar backbone and a similar secondary alcohol, which does not indicate developmental toxicity at high doses (1000 mg/kg bw). The 3000 mg/kg bw is considered an overdose also because readily oral absorption is anticipated.

Data matrix

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

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

The results presented above show that the analogues of CP Formate show no effects on fertility and developmental toxicity. For fertility the NOAEL is > 1000 mg/kg bw from the repeated dose toxicity of Applelide (the highest dose tested). The NOAEL for developmental toxicity is 1000 mg/kg bw, because body weight effects on pups were seen for CP Butyrate in the presence of decreased body weight gain of the dams.

This means that CP Formate does not need to be classified for fertility and developmental toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

The NOAEL of> 1000 mg/kg bw (highest dose tested) for fertility and the NOAEL of 1000 mg/kg bw for developmental toxicity indicates no effect at the limit dose prescribed by the OECD guidelines for testing. Therefore a DNEL derivation for these endpoints is not needed and the conclusion becomes “no hazard identified” for fertility and developmental toxicity.

Data matrix for CP Formate and the analogues for fertility and developmental 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
Repeated dose toxicity	Read across	>=1000 mg/kg bw (OECD TG 407)	>=3000 mg/kg bw (similar to OECD TG 407)
Repeated dose toxicity in dams			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
Fertility	Read across	No effects on male and female gonads were seen at >=1000 mg/kg bw, in an OECD TG 407	No effects on male and female gonads were seen at >= 3000 mg/kg bw, in a similar to OECD TG 407
Developmental toxicity	Read across	No data	No effects at 1000 mg/kg bw in a similar to OECD TG 414 test

# Molecular structures are presented 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.

Blackburn K, Bjerke D, Daston G, Felter S, Mahony C, Naciff J, Robison S, Wu S, 2011, Case studies to test: A framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments, Regul Toxicol Pharmacol., 60, 120-35

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 absence of adverse effects on reproductive organs in the repeated dose toxicity study with the structural analogue Applelide and based on the absence of developmental toxicity in the absence of maternal toxicity in the developmental toxicity study with the structural analogue CP Butyrate, classification is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.