3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-indenyl propionate

Endpoint summary

Description of key information

The repeated dose toxicity of Cyclaprop is based on data of two structural analogues: Cyclacet and Cyclobutanate

- Key study: Cyclacet 90-day rat NOAEL ≥1500 mg/kg bw/day (OECD 408)

- Supporting study: Cyclacet systemic toxicity in Reproductive/Developmental Toxicity screening study (OECD TG 421) >=1000 mg/kg bw.

- Supporting study: Cyclobutanate 28 -day rat NOAEL ≥ 1000 mg/kg bw (OECD 407).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

The 90-day oral repeated dose toxicity study and Reproscreen with Cyclacet and the 28-day oral repeated dose toxicity study with Cyclobutanate are adequate for covering this endpoint.

Additional information

For assessing the repeated dose toxicity of Cyclaprop information from Cyclacet is used. The latter substance has been tested according to the OECD TG 408 and OECD TG 421 of which the summaries are presented below. In addition, a repeated dose toxicity study with Cyclobutanate will be used to support the read-acoss. The summary of the experimental information available is presented first and thereafter the read across rationale.

Cyclacet 90 -day repeated dose study (OECD TG 408)

Introduction and method

In a sub-chronic repeated dose toxicity study performed according to OECD 408 and under GLP conditions, rats were orally exposed to Cyclacet for 90 days via the food. The dose levels were 0, 200, 2000, 6000, and 20000 ppm, corresponding to 0, 15.3, 154.9, 464.1, and 1504.6 mg/kg bw/day, respectively. Clinical signs, functional observations, body weight change, dietary intake, and water consumption were monitored during the study. During week 7 and at the end of the study, haematology, blood chemistry and urinalysis were evaluated. Furthermore, oestrus cycle assessment was performed on female animals between week 6 and 7 and week 12 and 13. Opthalmoscopic examination was performed at the start and end of the study in control and high-dose animals. At the end of the study, all animals were subjected to gross necropsy examination and histopathological examination of tissues was performed on animals from control and high-dose. Furthermore, sperm assessment was performed on males at necropsy.

Results

Clinical signs: Reduced overall body weight was observed in male and female animals in the highest dose group, correlated with reduced food consumption and adverse effects on food efficiency, indicating decreased food palatability rather than an adverse effect of the test substance.

Haematology: No effects were found.

Blood biochemistry: Blood chemical investigations showed reductions in chloride and sodium concentration of 1 -5%, which is considered minimal. The minimal decrease in ASAT and ALAT levels is not toxicological relevant. The reduction and increase in bile acids in males and females, respectively, is not considered toxicologically relevant either. The increases in cholesterol in males treated with either 20000 or 6000 ppm are most likely linked to the liver weight findings and the histopathological effects seen in this organ and can be considered of an adaptive nature.

Organ weights and histopathological findings: Relative liver weight is increased in males only up to 17% with slight adaptive and non-adverse centrilobular to midzonal hepatocellular hypertrophy in males only. Increase in adrenal weights, both absolute and relative to terminal body weights detected in males treated with 20000 ppm were correlated with histopathological findings of the adrenal gland. Microscopic examinations revealed increased incidence in vacuolation of the zona fasciculata in all treated males. This finding may be regarded as an adaptive effect occurring as a stress response to the physiological changes observed. Macroscopic examinations also revealed effects on the kidney such as an increase in kidney weights and hyaline droplets nephropathy in all treated males. The hyaline droplets can be directly linked to accumulation of the alpha-2-microglobulin, which is unique to the mature male rat, therefore, this finding is adverse for the male rat but has no relevance for other species including man. No other treatment-related effects were observed.

Conclusion: Under the conditions of the test no toxicologically significant effects were observed both in males and females. Therefore the NOAEL is at the highest dose tested, 20000 ppm or 1500 mg/kg bw/day.

Cyclacet tested in Reproscreen study (OECD TG 421)

In this reproduction / developmental toxicity screening test there was no systemic toxicity seen and the NOAEL of >= 1000 mg/kg bw was derived, see for details the reproductive toxicity section.

Cyclobutanate tested in 28 -day study (OECD TG 407)

Cyclobutanate has been tested for repeated dose systemic toxicity in accordance with OECD TG 407. Male and female rats were exposed to 0, 15, 150 or 1000 mg/kg bw/day of cyclobutanate for 28-days. Also two satellite groups were included that were allowed to recover for 14 days. Mortality, clinical signs, body weight, food consumption and organ weights were recorded. Hematology, clinical chemistry and urine analysis, gross pathology and histopathology were performed. An increase in salivation from day 6 onwards was observed in the high-dose group animals, recovery was apparent in the 14 day period after treatment. This effect is usually considered attributable to an unpleasant tasting or locally irritant formulation rather than an indication of systemic toxicity. In the mid-dose group females a significant reduction of body weight was observed in the last week of treatment. As no dose-relationship was observed, this finding was not considered of toxicological importance. No other clinical signs or body weight effects were seen. Also no effects were seen in haematology and biochemical chemistry. (Relative) Organ weights and macroscopy did not show adverse effects. In males, a higher incidence of globular accumulations of eosinophilic material in the tubular epithelium was observed in male rats of the high and mid-dose group (hydrocarbon nephropathy). Regression of this condition was seen in the 14 days of recovery. All other remaining morphological changes were commonly observed in laboratory rats and therefore not considered to be of toxicological importance. Under the conditions of this study, no adverse effects were observed in female rats up to the highest dose group. For males, hydrocarbon nephropathy was observed histopathologically in the 150 and 1000 mg/kg bw/day dose groups. Hydrocarbon nephropathy is a male-rat specific effect, which is not observed in human. For human risk assessment, this effect should therefore not be taken into account and the NOAEL is set at 1000 mg/kg bw, the highest dose tested.

The repeated dose toxicity of Cyclaprop using read across from Cyclacet and Cyclobutanate

Introduction and hypothesis for the analogue approach

Cyclaprop is a propyl ester attached to a tricyclodecenyl fused ring structure. 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. For assessing the repeated dose toxicity of Cyclaprop the analogue approach is selected because for two closely related analogues repeated dose toxicity information is available which can be used for read across.

Hypothesis: Cyclaprop has similar repeated dose toxicity compared to Cyclacet and Cyclobutanate resulting in a similar NOAEL, because the one methyl shorter chain analogue Cyclacet has not shown any repeated dose toxicity in the 90-day toxicity test (OECD TG 408) and the Reprocreen (OECD TG 421). In addition, the one methyl longer chain analogue Cyclobutanate has not shown any repeated dose toxicity in the 28-day toxicity tests (oral route: OECD TG 407). The information from these source chemicals Cyclacet and Cyclobutanate is sufficiently reliable and the chemicals are very similar in structure to read across towards Cyclaprop.

Available information: The source chemical Cyclacet has been tested in a well conducted 90-day repeated dose toxicity test (OECD TG 408, GLP) up to 1500 mg/kg bw and in a reproscreen test (OECD TG 421, GLP) up to 1000 mg/kg bw. In addition, for a second source chemical Cyclabutanate, a 28-day repeated dose toxicity test (OECD TG 407, GLP) is available in which up to 1000 mg/kg bw has been tested. All studies have a reliability score 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 repeated dose toxicity.

Purity / Impurities

Cyclaprop is a reaction mass containing a mixture of two very similar isomers (5yl and 6-yl). The impurities are all below 1%.

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. 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, which is presented below.

Analogue justification: For Cyclaprop the two nearest neighbours are used: Cyclacet with one methyl group shorter in the alkyl chain and Cyclobutanate with one methyl group longer in this alkyl chain and for both these sources repeated dose toxicity information was available.

Structural similarities and differences: The target and the source chemicals, the Cycla-esters, have a tricyclodecenyl fused ring structure with an unsaturated bond in the right ring. On the left ring an ester bond is attached with a short alkyl chain (≤C4). The alkyl chain of Cyclaprop (C3) is one methyl group longer than of Cyclacet (C2) and one methyl group shorter than of Cylobutanate (C4). These differences between the target and source chemicals are not expected to behave differently compared to the target organs because the alkyl side chains (propyl versus acetyl or butyl) are not expected to influence significantly the repeated dose toxicity these chemicals.

Toxico-kinetic: The source chemicals and the target chemicals indicate similar toxico-kinetic characteristics based on the similarity in chemical structure and physico-chemical properties. The molecular weight of the target chemical is 206, whereas those of the source chemicals are 192 and 220, respectively. They are all liquids. As can be expected Cyclacet has a slightly higher water solubility and a slightly lower log Kow in view of the one methyl group smaller chain length, while the water solubility of Cyclobutanate is somewhat lower and the log Kow somewhat higher compared to Cyclaprop. For the toxico-kinetic characteristics of all three chemicals these physical-chemical differences are expected to be minimal with respect to the absorption and distribution. Cyclaprop, Cyclacet, Cyclobutanate and similar esters will metabolise as presented in the figure 1 below. The available carboxylases in the gut and liver will break down the ester bond into the respective alcohol and acid. The rate of metabolisation will be similar between Cyclaprop, Cyclacet, and Cyclobutanate because the alkyl-chains are short and straight and thus no atoms are sterically hindering these carboxylases. The systemic toxicity will therefore result from the Cycla-alcohol and the acids. The toxicity of the metabolites acetic acid and butyric acid did not raise concern in the repeated dose toxicity tests and therefore the toxicity of the propionic acid will not raise concern either. Cycla-alcohol may be further metabolised or excreted as such. Butyric acid and propionic acid being natural components of the body will be consumed in the Krebs cyclus.

Fig. 1 The metabolization pathway of the Cycla-esters. The R-group can be a straight alkyl chain C2-C4 (the chemical structures can be seen in the read across document attached to the Endpoint summary of the repeated dose section in IUCLID section 7.5)

Repeated dose mode of action (MoA): Cyclaprop is expected to cause the same male rat specific alpha-hydrocarbon nephropathy as Cyclacet and Cyclobutanate do. The alpha-2u globulin is conjugated to the Cycla-alcohol besides glucuronation (see Toxico-kinetic section).

Similarities in results for toxicological endpoints between the target and the source chemical(s): In the data matrix a summary of the other toxicological data are presented to show that there are hardly toxicological differences between the target and both source chemicals, which result in a similar C&L. The acute oral and dermal toxicity data show limited acute toxicity. Cyclaprop (the target), Cyclacet and Cyclobutanate (the source chemicals) only show slight skin and eye irritation, which do not warrant C&L. The source and the target chemicals also show negative results in the Ames tests.

Uncertainty of the prediction: The predictions of the NOAEL of 1500 mg/kg bw for the target chemical for a 90-day repeated dose toxicity has a high certainty because of close similarity between the target and the source chemicals presented above. The one methyl group difference in the alkyl chain will not have an impact on the repeated dose toxicity. The available information from the source chemicals are high quality repeated dose toxicity and Reproscreen tests (Reliability 1). For Cyclaprop a NOAEL is derived of >=1000 mg/kg bw because this is the limit dose. The NOAELs of the sources do not need to be converted because the repeated dose toxicity is from the same Cycla-alcohol and a conversion would not change the Cyclaprop NOAEL.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix at the end of this section.

Conclusions for repeated dose toxicity

For Cyclaprop repeated dose toxicity study is not available but for two nearest neighbours such data is available, and these can be used to fill the data gap of Cyclaprop. 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, which is presented here.For Cyclacet no systemic toxicity was seen in a dietary 90-day repeated dose toxicity study (OECD TG 408) and an oral Reproscreen study (OECD TG 421). For Cyclobutanate a NOAEL of >= 1000 mg/kg bw is derived in an oral gavage OECD TG 407. Based on these analogue data for Cyclaprop also a NOAEL of 1500 mg/kg bw can be derived for sub-chronic repeated dose toxicity.

Final conclusion: Cyclaprop has a NOAEL of >=1000 mg/kg bw.

Data matrix for the read across to Cyclaprop from Cyclobutanate and Cyclacet for repeated dose toxicity

Common names	Cyclaprop	Cyclacet	Cyclobutanate
	Target	Key source	Supporting source
Chemical structures
Cas no 5-yl Cas no of the generic	- 68912-13-0	2500-83-6 54830-99-8	1361017-07-3 113889-23-9
EC number	272-805-7	911-369-0	441-420-8
Empirical formula	C13H18O2	C12H16O2	C14H20O2
REACH registration	Registered	Registered	Registered
Molecular weight	206	192	220
Physico-chemical data
Physical state	liquid	liquid	liquid
Melting point (^oC)	< -20	< -20	< -20
Boiling point (^oC)	263	247	275
Water solubility (mg/l)	57	186	11.5
Log Kow	4.4	3.9	4.48
Human health endpoints
Toxico-kinetics: metabolisation	Cycla-alcohol and propyl-acid	Cycla-alcohol and acetic acid	Cycla-alcohol and butyric acid
Acute oral tox (mg/kg bw)	>5000 OECD TG 401)	2750 (OECD TG 401)	>5000 (OECD TG 401)
Acute dermal tox (mg/kg bw)	>5000 (OECD TG 402)	>5000 (OECD TG 402)	>2000 (OECD TG 402)
Skin irritation	Not irritant (OECD TG 404)	Not irritant (OECD TG 404)	Not irritant (OECD TG 404)
Eye irritation	Not irritant (OECD TG 405)	Not irritant (OECD TG 405)	Not irritant (OECD TG 405)
Skin sensitisation	Not a sensitiser (Read across from Cyclobutanate)	Not a sensitiser (Read across from Cyclobutanate)	Not a sensitiser at 100% (OECD TG 406)
Genotoxicity – Ames test	Negative (OECD TG 471)	Negative (OECD TG 471)	Negative (OECD TG 471)
Repeated dose toxicity (mg/kg bw/day)	RA from Cyclacet	NOAEL >=1500 (OECD TG 408)	NOAEL >=1000 (OECD TG 407)
Reproductive toxicity including fertility and developmental toxicity – reproscreen study	Read across from Cyclacet	NOAEL >=1000 for both endpoints (OECD TG 421)

Justification for classification or non-classification

Based on the information above resulting in no adverse effects up to the limit dose >=1000 mg/kg bw, the test substance does not need to be classified for repeated dose toxicity according to EU CLP (EC 1272/2008 and its amendments).