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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Zenolide tested for gene mutation in bacteria (OECD 471, GLP): Not mutagenic .

Zenolide tested for in vitro cytogenicity, chromosomal aberrations (OECD TG 473): Negative

Zenolide tested for in vitro gene mutations in mammalian cells by means of read across from Habanolide (OECD TG 476): Negative

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In this section the experimental information on genotoxicity of Zenolide and Habanolide is presented first. Thereafter the read across justification is presented.

Zenolide: Ames

In a study, performed in accordance with GLP and OECD guideline 471, the test substance was examined for its possible mutagenic activity in the bacterial reverse mutation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and the Escherichia coli strain WP2uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The test substance was diluted in DMSO. Two independent mutation tests were performed. A top dose level of 5000 µg/plate, based on a preliminary test, was chosen for the first mutation study. Other dose levels used were a series of 2-fold dilutions of the highest concentration. A total of ten dose levels were used in the first test to ensure that sufficient non-toxic concentrations were assessed. Toxicity was observed towards all the strains at the top three dose levels, down to 625 µg/plate for TA1538 and down to 312.5 µg/plate for TA98 and TA100. For the second mutation test a top dose level of 12500 µg/plate was chosen and the bottom two dose levels not included for TA1535, TA1537 and WP2 uvrA. Toxicity was observed towards all the strains at the top dose level and down to 312.5 µg/plate for TA1538, TA98 and TA100. No evidence of mutagenic activity was seen at any dose level in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that the test substance shows no evidence of mutagenic activity in this bacterial system.

Zenolide: In vitro cytogenicity assay with mammalian cells

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to the test substance in DMSO both in the presence and absence of S9 mix (derived from Aroclor 1254 -induced rats). Solvent and positive control cultures were also prepared. After the appropriate incubation time, cell division was arrested using Colcemid, the cells were harvested and slides prepared. In order to assess the toxicity, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis: First test: Without S9 (3 hours treatment, 18 hours recovery): 200, 250 and 275 µg/ml; With S9 (3 hours treatment, 18 hours recovery): 200, 300 and 400 µg/ml. Second test: Without S9 (21 hours continuous treatment): 225, 250 and 275 µg/ml; With S9 (3 hours treatment, 18 hours recovery): 350, 500 and 600 µg/ml. In both the absence and presence of S9 mix, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the solvent control, in either test. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells. It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Habanolide: In vitro gene mutation assay with mammalian cells

In a mouse lymphoma assay (OECD guideline 476, GLP) L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. Three-hour exposures were used both with and without activation (S9) in Experiment 1. In Experiment 2 the exposure time without activation was increased to 24 hours. The dose range of test material was selected on the results of a preliminary toxicity test: 2.5 to 40 pg/ml in the absence of S9 and 5 to 80 pg/ml in the presence of S9 for the first experiment. For the second experiment the dose range was 10 to 70 pg/ml with S9 and 5 to 50 pg/ml without S9. The maximum dose level used was limited by the presence of precipitate. A precipitate of the test material was not observed at any dose level used in the main study. The vehicle (solvent) controls gave acceptable levels of mutant frequencies. The positive control treatments, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency. The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. In conclusion, Habanolide did not induce any statistically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non-mutagenic under the conditions of the test.

Supporting information from Musk T: In the ECHA disseminated dossier of Musk T, a HPRT test, performed according to OECD TG 476 and following GLP, is presented showing that Musk T is not mutagenic.

The read across justification is presented below

Zenolide(CAS #54982-83-1) and its genotoxicity in vitro in mammalian cells using read across from Habanolide (CAS #111879-80-2) with support from Musk T (CAS #105-95-3).

Introduction and hypothesis for the analogue approach

Zenolide is a cyclic aliphatic double ester for which there is information on gene mutations in bacterial cells and on cytogenicity in mammalian cells, but there is no information on gene mutations in mammalian cells. In accordance with Article 13 of REACH, lacking information can be generated by means other than experimental testing, i.e. applying alternative methods such as QSARs, grouping and read-across. For assessing the gene mutations in mammalian cells for Zenolide the analogue approach is applied. For the structural related analogue Habanolide such information is available and will be used for read across. Data on Musk T will be used to further support the analogue approach.

Hypothesis: Zenolide is negative in the in vitro gene mutation mammalian cells assay because Habanolide is negative in this test.

Available information: Zenolide has experimental information on gene mutations in bacterial cells (Ames) and on chromosomal aberrations (OECD TG 471 and 473, respectively). Habanolide has been tested for gene mutations in mammalian cells according to OECD TG 476. All tests are performed following GLP and considered to be Klimisch 1.

Target chemical and source chemical(s)

The information on Zenolide and Habanolide supporting the read across are presented in the Data matrix. To further support the read-across information on Musk T is included.

Purity / Impurities

Zenolide is a mono-constituent with a high purity> 95% and therefore the impurities are not expected to influence the results.

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 selection: When Zenolide was REACH registered in 2010, Habanolide was the most closely related structural analogue for which data on gene mutations in mammalian cells was available. Data on the more closely related Musk T could not be used, because the OECD TG 476 study was performed in 2013. However, for this read across justification, data on Musk T will also be taken into account as supporting information.

Structural similarities and differences: Zenolide and Habanolide are both cyclic aliphatic esters. They have a similar backbone and a similar functional group. The difference between these two structures is that Zenolide has a double ester while Habanolide has a single ester. In the cyclic-alkyl chain Habanolide has one unsaturated bond which is not present in Zenolide. Both differences are not anticipated to influence the genotoxicity.

Kinetic profile: Zenolide and Habanolide are expected to have similar absorption via all routes because of their similar structural and physico-chemical characteristics. Though the log Kow is somewhat lower and the water solubility somewhat higher for Zenolide compared to Habanolide, both substances are anticipated to be absorbed via all routes. Zenolide will be metabolised into Ethylene Glycol (CAS #107-21-1) and Dodecanedioic acid (CAS # 693-23-2), while Habanolide key metabolite will be 15-Hydroxypentadecanoic acid (CAS #4617-33-8).

Reactivity: The reactivity of Habanolide and Zenolide is considered similar based on their ester functionality. The double ester of Zenolide does not add to the genotoxic potential as can be seen in the Ames test and in the in vitro cytogenicity test. Also, the metabolites of Zenolide are not expected to have genotoxic reactivity as these structures do not have shown any genotoxic potential (ECHA disseminated dossiers on Ethylene Glycol and Dodecanedioic acid).

Uncertainty of the prediction: There are no remaining uncertainties other than those presented above. The absence of gene mutations in mammalian cells for Habanolide can also be used for Zenolide for the reasons presented above. This conclusion is substantiated by another analogue: Musk T, which has also a similar backbone and similar functional groups compared to Zenolide and is negative in the OECD TG 476 study (ECHA disseminated dossiers on Musk T).

Data matrix

The relevant information on physico-chemical properties and other environmental fate properties are presented in the Data Matrix below.

Conclusions on in vitro gene mutations in mammalian cells

For Zenolide a gene mutation test in bacteria (OECD 471) and an in vitro cytogenicity test in mammalian cells (OECD 473) is available, which are both negative. An in vitro gene mutation study in mammalian cells, however, is missing. For the structural analogue Habanolide such information is available. This information can be used for read across. When using read across the information should be accompanied with adequate and reliable documentation and this is presented in the current document. Habanolide is negative in the in vitro gene mutation assays in mammalian cells (OECD 476) and this can be applied to Zenolide. Final conclusion: Zenolide is negative for in vitro mutagenicity in mammalian cells.

 

Data matrix to support the read across to Zenolide (Target) from Habanolide (Key source) and Musk T (Supporting source) on in vitro mutagenicity in mammalian cells

Common names

Zenolide

Habanolide

Musk T

 

Target

Key Source

Supporting source

Chemical structures

CAS no

54982-83-1

111879-80-2

34902-57-3

105-95-3

EC no

259-423-6

422-320-3

203-347-8

Registration information

Yes

Yes

Yes

Empirical formula

C14H24O4

C15H26O2

C15H26O4

Molecular weight

256

238

270

Physico-chemical data

 

 

 

Physical state

Liquid

Liquid

Liquid

Water solubility (mg/l)

75

0.95

14.8

Log Kow

3.65

5.45

4.3

Human health

 

 

 

Gene mutations in bacterial cells (Ames)

Negative

(OECD TG 471)

Negative

(OECD TG 471)

(ECHA dissemination site)

Negative

(OECD TG 471)

(ECHA dissemination site)

Cytogenicity in mammalian cells

Negative

(OECD TG 473)

Negative

(OECD TG 473)

(ECHA dissemination site)

Negative

(Similar to OECD TG 474)

(ECHA dissemination site)

Gene mutations in mammalian cells

Negative

(Read across)

Negative

(OECD TG 476)

Negative

(OECD TG 476)

(ECHA dissemination site)

References:

Dodecanedioic acid, REACH dossier:https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/14886, site visited April 2019

Ethylene Glycol, REACH dossier:https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/15973, site visited April 2019

Musk T,REACH dossier:https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/14715, site visited April 2019

Justification for classification or non-classification

Based on the information presented above the substance is not mutagenic and therefore does not need to be classified according to EU CLP (EC 1272/2008 and its amendments).