Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

Endpoint summary

Description of key information

Repeated dose toxicity: 90-day rat NOAEL ≥ 1500 mg/kg bw/day (Read across from Cyclacet, OECD TG 408).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

For assessing the repeated dose toxicity of Cyclabute information from Cyclacet is used. Below a summary of the 90-day repeated dose toxicity study of Cyclacet is presented, the supporting 28-repeated dose of Cyclobutanate and the systemic toxicity in the reproscreen study (OECD TG 421) of Cyclacet. Thereafter the read across documentation is presented.

The key study selected is a 90 days subchronic repeated dose toxicity study on a closely related analogue Cyclacet.

In the sub-chronic repeated dose toxicity study with Cyclacet, performed according to OECD TG 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: 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. At the highest dose level, reduced chloride concentration, sodium concentration, aspartate aminotransferase levels, alanine aminotransferase levels, bile acid levels, and increased cholesterol levels were observed in male animals. The observed changes in aminotransferases, bile acid and cholesterol can be explained by the reduced food consumption. Changes in chloride and sodium concentrations may be explained by the observed kidney effects in males. Increased kidney weights were also observed in males, as well as hyalin droplet nephropathy, which is a rat-specific effect that is not relevant for humans. In females, no toxicologically significant effects were observed in clinical chemistry, organ weights or histopathology. Therefore, the NOAEL could be established as the highest dose tested, 20000 ppm or 1500 mg/kg bw/day, under the conditions of this test.

The supporting study is a 28-day oral repeated dose toxicity study on Cyclobutanate.

This test is conducted according to OECD guideline 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 urinanalysis, 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. 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 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 and male rats up to the highest dose group. For males, non-adverse hydrocarbon nephropathy was observed histopathologically in the 150 and 1000 mg/kg bw/day dose groups, which is an effect not relevant for humans. Therefore, a NOAEL of >=1000 mg/kg bw/day was established for female and male rats.

In addition to this key study absence of systemic effects was confirmed in the oral gavage reproductive toxicity screening study (OECD TG 421) of Cyclacet.

Overall conclusion: Based on these result, the NOAEL for Cyclabute for humans is established to be ≥1500 mg/kg bw/day based on the subchronic (90 -day) information from Cyclacet and with supporting subacute information from Cyclobutanate and the Reproscreen study of Cyclacet.

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

Introduction and hypothesis for the analogue approach

Cyclabute is an isobutyl 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 Cyclabute the analogue approach is selected because for two closely related analogue repeated dose toxicity information is available which can be used for read across. For Cyclacet a 90-day and for Cyclobutanate a 28-day oral repeat dose toxicity study is available. For Cyclacet also a Reproscreen study is available.

Hypothesis: Cyclabute has similar repeated dose toxicity compared to the Cyclacet and Cyclobutanate resulting in a similar NOAEL. This is based on similarity in chemical structure, bioavailability and Mode of Action. The two methyl longer side chain of Cyclabute compared to the analogue Cyclacet will not affect the outcome as is supported with the repeated dose toxicity information of Cyclobutanate.

Available information: The key source chemical Cyclacet has been tested in a well conducted 90-day repeated dose toxicity tests (OECD TG 408 under GLP) up to 1500 mg/kg bw. Also a Reproscreen test (OECD TG 421 under GLP) is available for Cyclacet in which up to 1000 mg/kg bw has been tested. In addition, for a second supporting substance, Cyclobutanate, a 28-day study is available according to OECD TG 407 under GLP.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemicals are shown in the data matrix below, including physico-chemical properties and toxicological information, thought relevant for repeated dose toxicity, of all substances.

Purity / Impurities The purity and impurities of the source chemicals do not indicate repeated dose toxicity potential. The impurities are all below < 10%. Also the purity and impurities of the target chemical do not indicate repeated dose potential and are below 10%. The unsaturated bond in the right ring can be at the 5-yl or the 6-yl position. This double bond has not been indicative for repeated dose toxicity and a change in position from 5-yl or 6-yl does not influence this.

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. It can also be used when the analogues used will have the same or similar metabolites. 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 selection: Cyclabute has the same backbone and the same ester functional group as its key (Cyclacet) and supporting (Cyclobutanate) analogues and for the key analogue repeated dose and fertility information is available fulfilling this Annex. For the supporting analogue supporting repeated dose toxicity information is available showing the repeated dose effects are the same whether the accompanying ester is acetate or a butanoate.

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 Cyclabute is two methyl groups longer than that of Cyclacet. Cyclobutanate has a straight chain while Cyclabute is branched. These differences between the target and source chemicals are not expected to behave differently compared to the target organs because the alkyl side chains (acetyl and butyl versus isobutyl) are not expected to influence significantly the repeated dose toxicity of 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 220, whereas those of the source chemicals are 192 and 220, for Cyclacet and Cyclobutanate, respectively. They are all liquids. Their vapour pressures are around 1 Pa (the measured value of Cyclobutanate is likely too high, because the estimated value is around 1 Pa too, using EpiSuite). It can be seen that the water solubility of the isobutanoic ester (Cyclabute) is somewhat lower and the log Kow somewhat higher compared to Cyclacet, which contains an acetic ester instead. 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. Cyclacet, Cyclabute and also Cyclobutanate and similar esters will metabolise as presented in the fig. 1 below. The available micro-organisms and/or carboxylases in the gut and liver will break down the ester bond into the respective alcohol and acid (see also toxico-kinetic section). The rate of metabolisation will be similar between the Cyclabute, Cyclacet and Cyclobutanate because the alkyl-chains are short and the branched methyl group in Cyclabute is not expected to sterically hinder this metabolisation. The systemic toxicity will therefore result from the Cycla-alcohol and the acid (Fig. 1). The toxicity of the metabolite butanoic-acid did not raise concern in the 28-day repeated dose toxicity and therefore the toxicity of the isobutanoic-acid will not raise concern either. This isobutanoic acid has been assessed at the WHO (1999) and it is expected that it will be metabolised into innocuous products. Cycla-alcohol will be glucuronidated and excreted as such.

Fig. 1 The metabolisation pathway of the Cycla-esters. The R group can be a straight or branched alkyl chain containing C2-C4 carbon atoms (see also 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 these substances show a low hazard profile resulting in the absence of C&L for human health effects. The effects seen are some increase in relative liver weight and alpha 2u-globulin hydrocarbon nephropathy in male rats.

Uncertainty of the prediction: The prediction of the NOAEL of >=1500 mg/kg bw for Cyclabute for subchronic toxicity has a high certainty because of the close structural similarity between Cyclabute and Cyclacet. The two methyl group differences in the alkyl chain will not have an impact on the repeated dose toxicity. This is supported using Cyclobutanate. In addition, all substances metabolise in the same alcohol. The respective acids are all closely related and expected to be metabolised into innocuous products. The available information from the source chemicals are high quality repeated dose toxicity test and Reproscreen tests (Reliability 1). Therefore the information can be used for read across without additional uncertainty.

Data matrix

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

Conclusions on hazard

For Cyclabute no repeated dose toxicity is available but for two analogues such information is available which can be used for read across. When using read across the result derived should be applicable for C&L and/or risk assessment. For Cyclacet (acetyl side chain) a well conducted 90-day repeated dose toxicity test is available (Reliability 1) with a NOAEL of >=1500 mg/kg bw. In addition, for Cyclobutanate no systemic toxicity was seen in a 28-day repeated dose toxicity study (OECD TG 407) at >=1000 mg/kg bw. For Cyclabute also a NOAEL of >= 1500 mg/kg bw can be derived for the endpoint sub-chronic repeated dose toxicity study and therefore classification is not needed.

Final conclusion: Cyclabute has a NOAEL of >=1500 mg/kg bw for sub-chronic exposure (90-days).

Data matrix for the read across to Cyclabute from Cyclacet and Cyclobutanate

Common names	Cyclabute (target)	Cyclacet (key source)	Cyclobutanate (supporting source
Chemical structures
CAS no	93941-73-2 (generic)	54830-99-8 (generic)	113889-23-9 (generic)
EC no	916-331-7	911-369-0	441-420-8
Molecular weight	220	192	220
Physico-chemical data
Physical state	liquid	liquid	liquid
Melting point, ^oC	< -20	< -20	< -20
Water solubility, mg/l	16	186	11.5
Log Kow	5.1	3.9	4.48
Human health endpoints
Toxico-kinetics: metabolisation	Cycla-alcohol and isobutanoic acid	Cycla-alcohol and acetic acid	Cycla-alcohol and butanoic acid
Acute oral tox in mg/kg bw	>10000 (eq OECD TG 401, using mice)	2750 (OECD TG 401)	>2000 (OECD TG 423)
Acute dermal tox in mg/kg bw	>2000 (based on acute oral toxicity information and read across from Cyclobutanate)	>2000 (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 (OECD TG 406)	Not a sensitiser (Read across from Cyclobutanate)	Not a sensitiser (OECD TG 406)
Genotoxicity – Ames test	Negative (OECD TG 471)	Negative (OECD TG 471)	Negative (OECD TG 471)
Repeated dose toxicity mg/kg bw	NOAEL >= 1500 (Read across from Cyclacet)	NOAEL >=1500 (OECD TG 408) NOAEL >= 1000 (OECD TG 421)	NOAEL >= 1000 (OECD TG 407)
Reproductive: fertility mg/kg bw	NOAEL >=1000 (Read across from Cyclacet)	NOAEL >=1000 (OECD TG 421) NOAEL >= 1500 (OECD 408 no effects on gonads)	(No effects seen on gonads in OECD TG 407)
Reproductive: developmental toxicity in mg/kg bw	NOAEL >=1000 (Read across from Cyclacet)	NOAEL >=1000 (OECD TG 421)	Read across from Cyclacet

* Vapour pressure is somewhat high also compared to the predicted value.

References

ECHA, 2010, Cyclacet registration under EC no: 911-369-0; https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/13281

OECD SIDS, 2003, SIDS Initial Assessment Profile of Isobutyric acid and Isobutyric anhydride, http://webnet.oecd.org/HPV/UI/handler.axd?id=26f9f920-1dc6-4e53-b5c4-fdd8b34af201

WHO, 1999, Evaluation of certain food additives, WHO Technical report series 885: http://whqlibdoc.who.int/trs/WHO_TRS_884.pdf     

 

Justification for classification or non-classification

Based on the NOAEL of >=1500 mg/kg bw/day, observed in a 90-day study of read across substance Cyclacet, Cyclabute does not need to be classified for (oral) repeated dose toxicity when considering the criteria outlined in EU CLP (1272/2008/EC and its updates).