2-tert-pentylcyclohexyl acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral combined repeated dose and reproductive screening study according to OECDTG 422: NOAEL ≥437 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline study performed with read across substance

Effect on fertility: via dermal route

Endpoint conclusion:

adverse effect observed

Additional information

The reprotoxicity of Coniferan is assessed by using information from Verdox. The Verdox information is summarised first and thereafter the read across justification is presented.

Verdox OECD TG 422 summary:

In a GLP compliant combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422, Wistar rats were treated with the test substance (0, 75, 200, and 500 mg verdox/kg bw/day, expected nominal dose levels) in the diet,during a premating period of 10 weeks and during mating (1 week), gestation and lactation until postnatal day 4. The actual test substance intake ranged between 46-69 for males and 42-71 for the females of the low dose group. For the mid dose group the actual test substance concentration ranged from 141-209 for the males and 129-209 for the females. For the high dose group the actual test substance concentration ranged from 415-617 for the males and ranged between 381-672 for the females. Clinical observations during the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. No toxicological relevant effects on body weight and food consumption were observed in both sexes. No treatment related effects were observed on pre-coital time, mating index, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of live pups on lactation. No treatment related clinical signs in pups, no effect on sex ratio in pups on day 1 and 4 of lactation, no effects on the mean pup body weight and body weight gain on lactation days 1 and 4 and no treatment-related macroscopic observations in pups that were stillborn or that died during lactation were observed. No effects on red blood cell variables or clotting potential was observed. A statistically significant increase in urea-concentration was observed in males of all dosing groups which might be related to the observed kidney effects (α2u-microglobulin nephropathy). No treatment-related effects were observed on sperm-parameters (epidydimal sperm motility, sperm count and morphology and testicular sperm count). An increased relative liver weight was observed in high dose males which was considered as an adaptive response to increased physiological demand. A dose related increase in relative kidney weight was observed in mid and high dose males which was related to the observed α2u-microglobulin nephropathy. No effects on organ weights were observed in females. Effects observed on kidney weights and urea-concentrations were considered to be related to α2u-microglobulin nephropathy which was confirmed by immunocytochemical staining of the α2u-microglobulin protein in the cortical tubular epithelial cells. This effect observed in rats is generally regarded as of no toxicological relevance for humans. Macrosopic and microscopic examination did not reveal any treatment-related effects. In conclusion, the NOAEL for Verdox for males and females in this study was ≥ 500 mg/kg body weight in the diet. Based on the absence of effects on fertility parameters and developmental parameters, the NOAEL for Verdox for fertility and developmental toxicity in this study was ≥ 500 mg/kg body weight in the diet. A NOAEL of 500 mg/kg body weight in the diet is equivalent to an overall intake of at least 505 mg/kg body weight/day for males and at least 437 mg/kg body weight/day for females.

Toxicity to reproduction of Coniferan (CAS #67874-72-0) using read across from Verdox (CAS #20298-69-5)

Introduction and hypothesis for the analogue approach

Coniferan is an acetate-ester attached to a cyclohexyl ring with a 2-methylbutan-2-yl group attached at the ortho-position. For this substance no data regarding fertility and developmental toxicity 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. For assessing the toxicity to reproduction of Coniferan the analogue approach is selected because for a closely related analogue, Verdox, fertility and developmental information is available which can be used for read across.

Hypothesis: Coniferan has similar fertility and developmental toxicity compared to Verdox as the additional methyl group attached to the tert-butyl-group in Coniferan is not expected to influence the fertility and developmental effects.

Available information: The target chemical Verdox is tested in an OECD TG 422 study. The test is well conducted and compliance with GLP guidelines and therefore receives a reliability of 1.

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 fertility and developmental effects of both substances.

Purity / Impurities

It is not expected that the impurities of the source chemical affect the read-across justification. Coniferan is a multi-constituent containing two stereo isomers. The purity of Coniferan is close to 100% therefore impurities will not affect fertility and developmental effects. In view of Verdox being a multi-constituent also containing two stereo isomers and having a purity close to 100%, there will be no significant impurities relevant for read across.

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.

Verdox was selected for read across because it was the most similar substance when compared to other cyclohexyl acetic esters such as presented in Belsito et al., 2008 (ECHA, 2017, RAAF).

Structural similarities and differences

Coniferan as well as the structural analogue Verdox have a cyclohexyl ring, with a tert-butyl group in ortho position and an acetate ester as a functional group. Coniferan has one additional methyl group attached to the tert-butyl group compared to Verdox. This one methyl group difference in this position is not expected to alter the reproductive toxicity of Coniferan.

Toxico-kinetic similarities and differences:

Absorption: Coniferan and Verdox have similar molecular weight and physico-chemical properties indicating similar absorption characteristics: both substances are liquids, molecular weights are 212.33 and 198.31 g/mol), log Kows are 5.4 and 4.75, the water solubilities are 7.6 and 10 mg/L, and vapour pressures are 4.24 and 9.72 Pa, respectively. This indicates that Coniferan is anticipated to have similar absorption characteristics compared to Verdox via the gastro-intestinal tract.

Metabolism:

Small chain straight alkyl esters such as Coniferan and Verdox are likely to be metabolized by carboxylesterases in the gut, liver, lungs and skin into the respective 2-methylbutan-2-yl-cyclohexanol and acetic acid and 2-tertbutyl-cylohexyl-alchol and acetic acid, respectively (Belsito et al., 2008, Yamada et al., 2013) (see metabolism figures 1 and 2). The alcohols will be conjugated and become more water soluble and will therefore more easily be excreted. Acetic acid is an endogenous component of the intermediary metabolism and will therefore not be assessed separately.

 

 

 

Fig. 1      The metabolisation pathway of the Coniferan and Verdox are presented, first and second structure, respectively. The Coniferan-alcohol and Verdox alcohol are the alcohol type of metabolites and acetic acid the other metabolite.

 Experimental data

A NOAEL of ≥ 505 mg/kg bw/day and ≥437 mg/kg bw/day for males and females, respectively, was observed as a result of repeated dosing in an OECD 422 study with Verdox. The NOAELs regarding fertility and developmental toxicity were also based on the information obtained with the OECD 422 study and were both ≥ 437 mg/kg bw/day as well. At the highest dose tested no adverse effects were seen.

Similarities in results for toxicological endpoints between the target and the source chemical(s)

In the data matrix it shows that for both substances acute oral toxicity was > 5000 mg/kg bw in rat. The similar absence of skin sensitisation properties indicates that both substances have a somewhat similar reactivity based on different testing regimes: Coniferan was tested in an LLNA, while for Verdox read across from other cyclic acetated was used.

Uncertainty of the prediction: There is no remaining uncertainty, in view of similarities in structure, toxico-kinetic profile and reactivity profile of Coniferan and Verdox. Therefore, read across is justified. In accordance with ECHA Read-Across Assessment Framework (RAAF) (2017) the read across would receive a score of 5.

Data matrix

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

Conclusions per endpoint for Hazard assessment and C&L.

For Verdox an OECD 422, GLP complaint study was conducted (reliability 1). Wistar rats were treated with the test substance (0, 75, 200, and 500 mg Verdox/kg bw/day, nominal expected dose levels) in the diet, during a premating period of 10 weeks and during mating (1 week), gestation and lactation until postnatal day 4. No treatment-related adverse effects were observed and subsequently the NOAEL for Verdox for males and females in this study was ≥ 500 mg/kg body weight in the diet. A NOAEL of 500 mg/kg body weight in the diet is equivalent to an overall intake of at least 505 mg/kg body weight/day for males and at least 437 mg/kg body weight/day for females.

Final conclusion on hazard and C&L

No adverse fertility effects are seen for Verdox in an extended for almost 90-days OECD TG 422, therefore for Coniferan no adverse fertility effects are anticipated. This means that the endpoint conclusion will be: No hazard identified. This also means that Coniferan is not classified for fertility toxicity.

No adverse developmental toxicity effects are seen for Verdox in an extended for almost 90-days OECD TG 422, therefore for Coniferan no adverse developmental toxic effects are anticipated. This means that the endpoint conclusion will be: No hazard identified. This also means that Coniferan is not classified for developmental toxicity

Classification and labelling is not needed for reproductive toxicity for this endpoint according to CLP Regulation (EC) No. 1272/2008 and its updates.

Data matrix for the read across to Coniferan and Verdox

Common names	Coniferan	Verdox
Chemical structures
CAS no	67874-72-0	20298-69-5
EC no.	Registration in 2018	243-718-1 (registered)
Empirical formula	C13H24O2	C12H22O2
Physico-chemical data
Molecular weight	212.33	198.31
Physical state	Liquid	Liquid at 30°C
Melting point, °C	-20	29.8
Boiling point, °C	251.9	232
Vapour pressure, Pa	4.24 at 24°C	9.72 at 23°C
Water solubility, mg/L	7.6 at 24°C	10 at 23°C
Log Kow	5.4	4.75
Human health endpoints
Toxico-kinetics: metabolisation	Coniferan is expected to be metabolised to 2-methylbutan-2-yl-cyclohexanol and acetic acid	Verdox is expected to be metabolised to 2-tert-butylcyclohexanol and acetic acid
Acute oral	Two studies similar to OECD 401. LD50 4388 mg/kg bw (mouse) LD50 > 5000 mg/kg bw (rat)	Similar to OECD 401. LD50 4600 mg/kg bw
Acute dermal	Similar to OECD 402 LD50> 5000 mg/kg bw	Similar to OECD 402 LD50 > 5000 mg/kg bw
Repeated dose (OECD TG 422)	Read across from Verdox	OECD 422 NOAEL ≥ 437 mg/kg bw/day
Reproductive toxicity	Read across from Verdox
Fertility (OECD TG 422)	Read across from Verdox	NOAEL ≥437 mg/kg bw
Developmental toxicity (OECD TG 422)	Read across from Verdox	NOAEL ≥ 437 mg/kg bw

 References

Belsito et al., A toxicologic and dermatologic assessment of cyclic acetates when used as fragrance ingredients, Food and Chemical Toxicology 46 (2008) S1–S27

Yamada et al., A category approach to predicting the repeated-dose hepatotoxicity of allyl esters, Regulatory Toxicology and Pharmacology 65 (2013) 189–195

Effects on developmental toxicity

Description of key information

Developmental toxicity for Coniferan using information of Verdox that is tested according to OECD TG 422: No adverse effects observed: NOAEL ≥437 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline study performed with read across substance

Additional information

The developmental toxicity of Coniferan is assessed by using information from Verdox. The Verdox information is summarised first and thereafter the read across justification is presented.

Verdox OECD TG 422 summary: In a GLP compliant extended combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422, Wistar rats were treated with the test substance (0, 75, 200, and 500 mg verdox/kg bw/day, expected nominal dose levels) in the diet,during a premating period of 10 weeks and during mating (1 week), gestation and lactation until postnatal day 4. The actual test substance intake ranged between 42-71, 129 -209, and 381-672 for the low, mid, and high dose group, respectively. Clinical observations during the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. No toxicological relevant effects on body weight and food consumption were observed. No treatment related effects were observed on pre-coital time, mating index, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of live pups on lactation. No treatment related clinical signs in pups, no effect on sex ratio in pups on day 1 and 4 of lactation, no effects on the mean pup body weight and body weight gain on lactation days 1 and 4 and no treatment-related macroscopic observations in pups that were stillborn or that died during lactation were observed. No effects on red blood cell variables or clotting potential was observed. No effects on organ weights were observed in females. Macrosopic and microscopic examination did not reveal any treatment-related effects. In conclusion, the NOAEL for maternal toxicity in this study was ≥ 500 mg/kg body weight in the diet. Based on the absence of effects on developmental parameters, the NOAEL for Verdox for developmental toxicity in this study was ≥ 500 mg/kg body weight in the diet. A NOAEL of 500 mg/kg body weight in the diet is equivalent to an overall intake of at least 437 mg/kg body weight/day for females.

Justification for classification or non-classification

Based on the absence of adverse effects on fertility and of developmental toxicity at the highest dose tested in the oral (diet) combined repeated dose toxicity study with reproduction / developmental toxicity screening test in rats, classification of Coniferan for effects on fertility or developmental toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its updates.