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Key value for chemical safety assessment

Additional information

Note to the reader: Please refer also to the documentation in the endpoint study record to Exp_Key (CCP-V-1).003

1.   Executive Summary

According to REACH Annex XI, grouping of substances is used as read across to fulfil the data requirement according to REACH Annex VIII, 8.4.2 (In vitro cytogenicity study in mammalian cells or in vitro micronucleus study) for CCP-V-1 ((trans(trans))-4'-vinyl-4 -(4 -methylphenyl)bicyclohexyl, CAS No. 155041 -85 -3).

A category of seven chemically similar liquid crystal compounds is built. The chemical similarity is based on a common core structure consisting of a bicyclohexyl moiety linked to a phenyl group. The structural variations of the category compounds encompass partial fluorination of the phenyl ring and modifications of the alkyl- or alkenyl- substitutions at the para-position of the phenyl- and cyclohexyl ring. All category members show similar physicochemical properties, i.e. low water solubility and high logPOW value resulting in a very limited bioavailability. For all seven members of the category, in vitro mammalian cytogenicity tests according to OECD 473 and GLP-requirements are available (reliability score according to Klimisch: 1). None of the compounds showed any indication for chromosomal damage in any of the tests. Thus, there is clear evidence that the category compounds are not clastogenic.

The query compound CCP-V-1 fulfils the category criteria for the chemical applicability domain, the physiochemical space and the biological response in the mutagenicity assays. CCP-V-1 shares the core structure with the category. Both substituents, the alkenyl group at the cyclohexyl ring as well as the methyl group at the phenyl ring are present in one or more members of the category, and have, thus, been tested in in vitro cytogenicity tests. Based on these data the category provides sufficient and reliable information to fill the data gap for the cytogenicity assay, i.e. chromosomal aberration, for CCP-V-1. Furthermore, CCP-V-1 was negative in the mammalian gene mutation assay (MLA) (OECD 476) performed with colony sizing, thus, adding supplementary evidence for the absence of any possible clastogenic potential.

In conclusion, there is sufficient and reliable evidence from the chemical category and for CCP‑V‑1 itself to conclude that CCP-V-1 is not genotoxic, i.e. not clastogenic. Therefore the data requirement according to REACH Annex VIII, 8.4.2 is fulfilled and no further experimental testing is considered justified.

 

2.   Introduction

Under REACH, testing requirements for individual substances are based on the specific information requirements laid down in Annexes VI-X.

Annex XI of REACH describes general rules for the adaptation of the testing regime of Annex VII-X. In chapter 1.5 of Annex XI, data gap filling based on read across and grouping of substances is explained. Data gap filling by a grouping approach requires substances with structural, physicochemical or toxicological properties similar to those of the query compound. The similarity may be based on:

·        a common functional group

·        common precursors or breakdown products resulting in structurally similar chemicals

·        a constant pattern in changing of the potency of certain properties across the category

This paper provides the reasoning on the grouping of substances in a category that serves to fulfil the data requirement according to REACH Annex VIII, 8.4.2 (in vitro cytogenicity study in mammalian cells or in vitro micronucleus study) for CCP-V-1 ((trans(trans))-4'-vinyl-4-(4-methylphenyl)bicyclohexyl, CAS No. 155041-85-3).

 

3.   Category definition and its members

3.1. Category Definition

3.1.1.  Category Hypothesis

This category covers a group of structurally similar liquid crystalline compounds sharing a common core fragment consisting of a bicyclohexyl moiety linked to a phenyl group.

 

For details concerning the structural elements, please refer to Figure 1 of the documentation in the endpoint study record Exp_Key (CCP-V-1).003.

The structural variations encompass partial fluorination of the phenyl ring, the length of the alkyl side chain, i.e. an alkyl or alkenyl group, and the terminal substituent at the phenyl ring, which may either be fluorine, alkyl, alkoxy or trifluoromethoxy groups. The category currently comprises seven liquid crystalline compounds. Besides the structural similarity these compounds also show similar physicochemical properties, i.e. low water solubility and a high logPOWvalue, thus indicating poor bioavailability.

All category members have been investigated in GLP compliant mutagenicity assays in vitro according to OECD guidelines 471 and 473 (Klimisch score for reliability: 1).

All compounds in this category are non-mutagenic in the bacterial reverse mutation test (Ames-Test). Furthermore, in vitro studies in mammalian cells have clearly demonstrated that all compounds of this category are negative in the in vitro chromosomal aberration test, i.e. are not clastogenic. In addition, for one category member (no. 5) and the query compound CCP-V-1 in vitro mammalian cell gene mutation tests (mouse lymphoma test) with colony sizing did neither indicate any mutagenic nor clastogenic potential for both compounds.

 

3.1.2.  Applicability Domain

The category applies to liquid crystalline compounds containing a bicyclohexyl-phenyl fragment as core structure (Figure 1) that contains terminal alkyl, alkoxy or alkenyl substitutions at the cyclohexyl or phenyl moiety. The phenyl ring can be partially fluorinated.

 

3.1.3.  List of endpoints covered

The category applies to the following endpoints:

-     n-Octanol/water partition coefficient (log Pow)

-     Water solubility

-     Mutagenicity and cytogenicity

3.2. Category members

Table 1 summarizes the structural formulas, chemical names and CAS numbers of the category members. The first row (substance 1) shows the query molecule, CCP-V-1, and following rows (substances 2-8) depict the category members.

 

Cpd.
No.

Structure

Chemical Name

CAS

1

1-[4-(4-ethenylcyclohexyl)cyclohexyl]-4-

methylbenzene

155041-85-3

2

1-methyl-4-[4-(4‑propylcyclohexyl)cyclo-

hexyl]benzene

84656-75-7

3

4-[4-(4-ethenylcyclohexyl)cyclohexyl]-1,2-

difluorobenzene

142400-92-8

4

1-[4-(4-ethylcyclohexyl)cyclohexyl]-2,3-difluoro-4-

methylbenzene

174350-08-4

5

1,2,3-trifluoro-5-[4-(4-

propylcyclohexyl)cyclohexyl]benzene

131819-23-3

6

2,3-difluoro-1-methoxy-4-[4-(4-

propylcyclohexyl)cyclohexyl]benzene

431947-34-1

7

1-ethoxy-2,3-difluoro-4-[4-(4-

propylcyclohexyl)cyclohex-1-en-1-yl]benzene

124728-62-7

8

1-[4-(4-propylcyclohexyl)cyclohexyl]-4-

(trifluoromethoxy)benzene

133937-72-1


Table 1. Chemical names and CAS numbers of the target molecule (CCP-V-1, compound 1) and the category members (compounds 2-8). For chemical structures, please refer to the attached endpoint study record Exp_Key.003.

 

3.3. Purity

The purity of all compounds of the category exceeds 99.5 % as investigated by chromatographic methods.


 

4.   Category justification

The category compounds are liquid crystalline molecules with a bicyclohexyl-phenyl core structure (see table 2 and figure 1). All category members have a low water solubility (< 1 mg/L), a molar mass between 280 and 400, and a high octanol-water partition coefficient (logPOW> 4.5) (see table 3). Their behaviour in water and biological systems is dominated by their hydrophobic character leading to a very limited bioavailability. Experimental physicochemical and in vitro mutagenicity (Ames test and chromosome aberration) data exist for all category members. All category members were non-mutagenic in the Ames test and not clastogenic in thein vitrochromosome aberration test in mammalian cells. In conclusion, compounds of bicyclohexylphenyl category have no mutagenic or clastogenic potential.

(trans(trans))-4'-vinyl-4-(4-methylphenyl)bicyclohexyl (CCP-V-1) is sharing the structural features presented in this category and shows the same physicochemical patterns. CCP-V-1 is not mutagenic in the Ames test and not mutagenic in thein vitromutagenicity test in mammalian cells (mouse lymphoma TK+/- assay).

The overlap in structural space (table 2), in physicochemical properties (table 3), and in biological data (table 4) clearly demonstrates that CCP-V-1 perfectly meets the criteria for this category and that the data obtained for the category compounds can thus be used for read across to CCP-V-1.

The category approach considers structural and physicochemical similarity together with a constant pattern in toxicological data and is thus fully compliant with REACH Annex XI and further criteria as defined in the REACH Guidance Chapter R.6.


 

5.   Data matrix for the category

5.1. Chemical structures

Table 2 summarizes the chemical structures for the category compounds (No. 2-8). The header column shows the structural fragments of CCP-V-1. In each column the presence (+) or absence (-) of the different structural fragments of CCP-V-1 for each of the category members is given.

 

Cpd. No


CCP moiety


vinyl moiety


methyl group

2

+

-

+

3

+

+

-

4

+

-

+

5

+

-

-

6

+

-

-

7

+

-

-

8

+

-

-


Table 2. Structural fragment analysis of the category compounds (2-8) with respect to the major structural features of the query structure. For chemical structures, please refer to the attached endpoint study record Exp_Key.003.

 

The core fragment of CCP-V-1, i.e. the bicyclohexyl-phenyl moiety (see column 1), is present in all category members (compounds 2-8). This structural feature is mainly responsible for the physicochemical profile (see chapter 3.2), i.e. low water solubility and low bioavailability, of this category.

The alkenyl substitution at the cyclohexyl moiety (column 2) is covered in one category molecule (compound 3), while the terminal methyl substituent (column 3) is present in two category members (compounds 2 and 4). As can be seen from the data provided (see chapter 3.2 and 3.3), these functional groups have no relevant impact on the physicochemical and biological profile observed for this category.

In conclusion, all structural fragments of CCP-V-1 are present in one or more members of the category. Thus, CCP-V-1 is completely represented by the category.

 

5.2. Physicochemical data

Table 3 summarizes the physicochemical data for the category compounds and the query compound, CCP-V1.

 

Cpd.
No.

 

M 
[g/mol]

logPOW

Experimental/
Calculated*

logPOW> 4.5

Water Solubility
[mg/L]

Experimental/
Calculated*

Water solubility
< 1 mg/L

(< Limit of Detection, LOD)

1


(CCP-V-1)

282.47

> 4.7

+

< 0.005

+

2

298.51

> 4.7

+

0.0006

+

3

304.42

> 4.7*

+

< 0.01*

+

4

320.47

> 5.7

+

< 0.5

+

5

338.46

> 5.7

+

< 0.5

+

6

350.49

> 6.5

+

< 0.0022

+

7

362.50

> 6.5

+

< 0.04

+

8

368.48

> 5.7

+

< 0.2

+

 

Table 3. Comparison of the physicochemical data of the category compounds and the query compound CCP-V1. For chemical structures, please refer to the attached endpoint study record Exp_Key.003.

 

All category members (compounds 2-8) as well as the query compound (CCP-V-1) share common physicochemical properties, i.e. molar mass range, octanol-water partition coefficient and water solubility. The molar mass of all compounds is between 280 and 400 g/mol. The logPOWvalues are higher than of 4.5. All compounds are poorly water soluble (< 1 mg/L). For poorly soluble compounds, it is difficult to exactly determine log POW and water solubility experimentally because the concentrations in water are far below the limits of detection of the available analytical methods. Therefore, no exact solubility data and logPow are available for these compounds. The limit of detection (LOD) of the individual analytical method is given as water solubility limit in the table.

 

5.3. Mutagenicity data

All category compounds as well as the query compound (CCP-V-1) have been characterized in various GLP compliant mutagenicity assays. In detail, the assays performed cover the Ames bacterial mutagenicity assay (OECD TG 471), mammalian gene (tk+/-) mutation assay (OECD TG 476), and mammalian cytogenicity assay (OECD TG 473). Table 4 summarizes the results obtained in the mammalian cell assays for each category compound and the query compound (CCP-V-1).

 

Cpd. No

 

Mammalian Cytogenicity

Mammalian Gene Mutation

1

(CCP-V-1)

 

negative

2

negative

 

3

negative

 

4

negative

 

5

negative

negative

6

negative

 

7

negative

 

8

negative

 


Table 4. Summary of in vitro mammalian cell mutagenicity data. For chemical structures, please refer to the attached endpoint study record Exp_Key.003.

 

The category members (compounds 2-8) have been characterized in the bacterial reverse mutation assay (Ames) and mammalian cell cytogenicity assay (chromosomal aberration test, CA). All of them are negative in these tests clearly demonstrating the absence of any mutagenic and clastogenic potential for this category. In addition, one category member (compound 5) and the query compound CCP-V-1 were tested in a mammalian cell gene mutation assay according to OECD TG 476 (mouse lymphoma tk+/- assay, MLA) including colony sizing.

There is overwhelming evidence that the mouse lymphoma assay has the ability to detect both gene mutations and chromosomal damage (OECD guideline 476; Mueller et al., 1999). There is also considerable evidence that spontaneous and induced small colony tk-/-mutants in the mouse lymphoma assay carry mainly large deletions or chromosomal rearrangements (Clive et al., 1995; Sofuni et al., 1997; El-Tarras et al., 1995; Hozier et al., 1982; Hozier et al., 1985). Published data suggest that small and large colony populations very often represent the induction of chromosomal aberrations (e.g. large deletions) and gene mutations, respectively (Combes et al., 1995). It has been demonstrated that with appropriate test protocols the variousin vitrotests for chromosomal damage and the mouse lymphoma assay yield results with a high level of congruence for compounds that are regarded as genotoxic but yield negative results in the bacterial reverse mutation assay. Therefore, these systems are currently considered interchangeable when used together with other genotoxicity tests in a standard battery for genotoxicity testing of pharmaceuticals (ICH, 2011; FDA, 2000; FDA, 2006; Mueller et al, 1999) provided that colony sizing i.e. quantification of both, small and large mutant colonies, is performed (OECD guideline 476). The negative result for the category member (compound 5) in the mammalian gene mutation assay is in perfect agreement with the negative data in the chromosomal aberration test obtained for this category compound.

 

6.   Conclusion

The query compound CCP-V-1 fulfils the category criteria for the chemical applicability domain, the physiochemical space and the biological response in the mutagenicity assays. CCP-V-1 shares the core structure with the category. Both substituents, the alkenyl group at the cyclohexyl ring as well as the methyl group at the phenyl ring are present in one or more members of the category, and have, thus, been tested in in vitro cytogenicity tests. Based on these data the category provides sufficient and reliable information to fill the data gap for the cytogenicity assay, i.e. chromosomal aberration, for CCP-V-1. Furthermore, CCP-V-1 was negative in the mammalian cell gene mutation assay (MLA) (OECD 476) performed with colony sizing, thus, adding supplementary evidence for the absence of any possible clastogenic potential.

In conclusion, there is sufficient and reliable evidence from the chemical category and for CCP‑V‑1 itself to conclude that CCP-V-1 is not genotoxic, i.e. not clastogenic. Therefore the data requirement according to REACH Annex VIII, 8.4.2 is fulfilled and no further experimental testing is considered justified.


7.  References

Clive D, Bolcsfoldi G, Clements J, Cole J, Monma M, Majeska J, Moore M, Mueller L, Myhr B, Overbly T, Oudelhkim M-C, Rudd C, Shimada H, Sofuni T, Thybaud V, Wilcox P (1995) Consensus agreement regarding protocol issues discussed during the mouse lymphoma workshop, Portland OR, May 7, 1994. Environ. Mol. Mutagen. 25: 165-168

Combes RD, Stopper, Caspary WJ (1995) The use of the L5178Y mouse lymphoma cells to assess the mutagenic, clastogenic and aneugenic properties of chemicals. Mutagenesis 10: 403-408

El-Tarras A, Dubins JS, Warner J, Hoffman C, Cobb RR (1995) Molecular analysis of the TK locus in L5178Y large and small colony mouse lymphoma cell mutants induced by hycanthone methanesulfonate. Mutation Research 332: 89-95

FDA (2000) Redbook 2000: Toxicological Principles for the Safety Assessment of Food Ingredients, IV.C.1. Short-Term Tests for Genetic Toxicity. Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, Department of Health and Human Services, U.S. Food and Drug Administration, July, 7, 2000   
(
http://www.fda.gov/downloads/Food/GuidanceRegulation/UCM222779.pdf) (page 2, Recommended Tests)

FDA (2006) Guidance for Industry: Recommended Approaches to Integration of Genetic Toxicological Study Results (Draft November 2004). Center for Drug Evaluation and Research (CDER), Department of Health and Human Services, U.S. Food and Drug Administration (http://www.fda.gov/ohrms/dockets/98fr/04d-0493-gdl0002.pdf)

Hozier J, Sawyer J, Clive D, Moore M (1982) Cytogenetic distinction between the TK+ and TK- chromosomes in the L5178Y TK+/- -3.7.2C cell line. Mutation Research 105: 451-456

Hozier J, Sawyer J, Clive D, Moore M (1985) Chromosome 11 aberrations in small colony L5178Y TK-/- mutants early in their clonal history. Ann. N.Y. Acad. Sci. 107: 423-425

ICH (2011) S2(R1): Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), step 4, adopted 9 Nov 2011 (http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S2_R1/Step4/S2R1_Step4.pdf)

Mueller L, Kikuchi Y, Probst G, Schechtman L, Shimada H, Sofuni T, Tweats D (1999) ICH-Harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact. Mutation Research 436: 195-225

Sofuni T, Wilcox P, Shimada H, Clements J, Honma M, Clive D, Green M, Thybaud V, San RHC, Elliot BM, Mueller L (1997) Mouse lymphoma workshop, Victoria, British Columbia, Canada, March 27, 1996, Protocol issues regarding the use of the microwell method of the mouse lymphoma assay. Environ. Mol. Mutagen 29: 434-438

 




Short description of key information:
Ames test: negative
MLA assay in vitro: negative
CA assays from a group of similar compounds: negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Mutagenicity data are available from two experimental in vitro assays as well as from read across to structurally similar comounds. CCP-V-1 was studied in two different in vitro assays including the bacterial reverse mutation assay and the in vitro mammalian gene mutation assay (TK test). In these tests the test material did not show mutagenic/clastogenic potential. In addition, a range of structurally similar compounds was tested for the induction of chromosome aberrations. No indication for the induction of structural or numerical aberrations was detected.