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Description of key information

Verdox tested in a similar to Buehler test (OECD TG 406): non sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Limitations: amount of substance applied and time between challenge and scoring are unknown
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
The protocol looks similar to the Buehler test
Principles of method if other than guideline:
The guinea pigs were treated twice daily for 21 days on the shaved flank for the irritation portion. Following a 5 day rest, a virgin site was shaved on the neck and the test article applied for 5 consecutive days.
GLP compliance:
no
Type of study:
Buehler test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Buehler test has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Species:
guinea pig
Strain:
other: Pirbright white
Sex:
not specified
Details on test animals and environmental conditions:
- Weight at study initiation: ca. 300-500 g
Route:
other: epicutaneous (not specified)
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Concentrated form
Route:
other: epicutaneous (not specified)
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Concentrated form
No. of animals per dose:
20 (sex unknown)
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 42
- Exposure period: 21 days
- Test group: 20 animals
- Control group: none
- Site: shaved flank
- Frequency of applications: twice daily
- Duration: not specified
- Concentrations: concentrated form

B. CHALLENGE EXPOSURE
- No. of exposures: 5
- Day(s) of challenge: starting from 15 days post-exposure daily
- Exposure period: 5 days
- Test group: 20 animals
- Control group: none
- Site: shaved neck
- Concentrations: concentrated form
- Evaluation (hr after challenge): no data

Challenge controls:
No data
Positive control substance(s):
not specified
Key result
Reading:
1st reading
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No skin reactions were observed.
Group:
negative control
Remarks on result:
not measured/tested
Group:
positive control
Remarks on result:
not measured/tested

The dose level is assumed to be 100 % but the % is limitedly documented in the report.

Interpretation of results:
other: Not sensitising
Remarks:
in accordance with EU CLP (1272/2008 and its updates)
Conclusions:
The substance gave negative results in a Buehler study with guinea pigs (OECD TG 406).
Executive summary:

Skin sensitizing properties of Verdox were tested in the study with 20 Pirbright white guinea pigs. The study preceeded OECD guidelines and GLP but is similar to OECD TG 406. The study is a limitedly reported: amount of substance applied and time between challenge and scoring are unknown). Positive controls were not reported. The guinea pigs were treated twice daily for 21 days on the shaved flank for the irritation portion. Following a 5 day rest, a virgin site was shaved on the neck and the test article applied for 5 consecutive days. The length of the observation period was not specified. No skin reactions were noted in any of the animals.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitisation of Verdox was tested in a Buehler test of which the executive summary is presented below. The executive summaries of the HRIPT test with Verdox are presented in the study records. To support the absence of skin sensitisation of Verdox SAR and analogue information is presented thereafter.

Verdox sensitisation in the Buehler test

Skin sensitizing properties of Verdox were tested in the study with 20 Pirbright white guinea pigs. The study preceeded OECD guidelines and GLP but is similar to OECD TG 406. The study is a limitedly reported: amount of substance applied and time between challenge and scoring are unknown). Positive controls were not reported. The guinea pigs were treated twice daily for 21 days on the shaved flank for the irritation portion. Following a 5 day rest, a virgin site was shaved on the neck and the test article applied for 5 consecutive days. The length of the observation period was not specified. No skin reactions were noted in any of the animals.

Verdox and its non-sensitising properties using a Buehler test and supporting in silico information

Introduction and hypothesis for the read across approach

For Verdoxin vivo skin sensitisation data are available which is somewhat limited reported and/or concentrations are used below 100%. Therefore additional information is used to present additional information on the skin sensitisation potentialHRIPT information, QSARs, grouping and read-across.

Verdox is an acetate-ester of α-tert-butyl-substituted cyclohexanol.

Hypothesis:Verdox is not expected to have sensitizing properties based on in vivo testing information of the substance itself, SAR information considering the protein binding affinity and analogue information.

Available information: For Verdox a Buehler test has been performed (similar to OECD TG 406, without GLP, in 1972) on the pure substance. The study is, however, limitedly reported. Also a Human Repeat Insult Patch test (HRIPT) with Verdox is available, using 2 application sites on one individual (therefore there are 2 entries in IUCLID). The test concentration was 10% at both sites, which may be considered too low concentration for REACH purpose in absence of skin irritating properties.

Source chemical(s):The information on Verdox, the SAR and the analogue information is presented in the data matrix presented in Table 1.

Purity / Impurities: Verdox has a cis- and trans-form. These cis- and trans-forms of Verdox are not expected to influence the reactivity of the ester bond and thus its skin sensitising properties. It is not expected thatany impurities present (< 0.1%) in the target will significantly affect the skin sensitisation potential either.It can therefore be concluded that such impurities will not affect the assessment.

Skin sensitisation supporting information:

Theory: For skin sensitization two main causes for induction are known. Skin sensitisation is caused by covalent binding between the negatively charged skin proteins and the positively charged carbon-atom in the molecular structure due to adjacent electrophilic substructures such as oxygen atoms. This covalent binding between these two is the onset of an immunological reaction as presented in ECHA’s R.7.3.4.1. This onset can also be caused by radicals. Radical formation may be caused by hydroperoxides and peroxides: R-O-OH bonds or R1-O-O-R2 bonds, respectively.

In silico information: For Verdox the carbon-ester would be the potential site for attack for proteins in the skin. Due to the limitedly positively charged carbon the proteins will not bind (sufficiently) to cause an immunological reaction. This is because of the absence of additional electrophilic activating groups (such as a double bond or a halogen which increases the positive charge on the carbon-ester) on the alpha or beta position of the ester e.g. no halogens and no double or triple bonds. The SCCS has predicted the absence of sensitisation potential for Verdox (SCCS, 2012:http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_092.pdf). The OECD Toolbox and Derek Nexus both predict the absence of skin sensitisation for Verdox. They also predict the absence of hydroperoxide metabolites when using the skin metabolism profiler, which may be another cause for skin sensitisation. However, both these programs focus on the presence of structural alerts for skin sensitisation and less so on the absence. Therefore some skin sensitisation information from analogues is presented to further confirm the absence of skin sensitisation information.

Analogue information: The information of the analogues is summarised in the data matrix below. The structural similarities and differences between Verdox and the analogues are addressed below.

Structural similarities and differences: Verdox and the analogues presented all belong to the cyclic-esters. They all have a cyclohexyl-ring with short alkyl groups on different positions of the ring. The ester bond is the functional group and its reactivity is low because there are no electron-withdrawing groups closely attached to this ester (at alpha or a beta position) and no (hydro)peroxide structures are present in the structure or can be formed upon skin metabolism. The structural difference between Verdox and the analogue is the tert-butyl vs. the tert-pentyl group, isopropyl group and the position on the ring. These groups as such limitedly influence the reactivity of the ester bond because they are not electrophilic.

Bioavailability: Verdox and the cyclic-esters are all liquids and have somewhat similar log Kow values indicating that these substances will be absorbed by the skin to a similar extent.

Reactivity: The 2-position of the tert-butyl (Verdox) and pentyl group (Coniferan) especially in the cis-form will hinder the skin protein attack somewhat more compared to the 4-position of the isopropyl group, which would make this analogue more susceptible for attack compared to Verdox and presenting a conservative approach. The ethyl groups between the ester and cyclohexyl ring in the last two source chemicals are not expected to influence the reactivity of the ester bond because these groups are not electrophilic either.

Remaining uncertainties:There are no remaining uncertainties because the skin sensitisation mechanisms are well known and are well described by e.g. Aptula and Roberts (as referenced in ECHA’s R7.3.4.1). The experimental data of Verdox, the SAR and analogue information (especially Coniferan) all show the absence of skin sensitisation and therefore there are no remaining uncertainties.

Conclusion for skin sensitisation

Verdox is not a skin sensitiser. This is based on in vivo information for Verdox (Buehler test and HRIPT), using structural activity relationships (absence of structural alerts and limitedly active electrophilic substructures) and analogue information of which Coniferan is the most similar.

Table 1:    Information on Verdox, SARs and analogues important for assessment of skin sensitising properties.

Chemical name

2-tert-butylcyclo-hexyl acetate

(Verdox)

2-tert-pentylcyclo-hexyl acetate

(Coniferan)

4-Isopropylcyclo-hexyl propionate

1-(3,3-Dimethylcyclohexyl)ethyl acetate

Cyclohexylmethyl-carbinyl acetate

Veilex 2

 

Target

Source

Source

Source

Source

CAS

20298-69-5

67874-72-0

63449-95-6

25225-10-9

13487-27-9

Molecular structure

EINECs

243-718-1

267-500-0

264-165-2

246-737-3

236-798-4

REACH registration

Registered

Registered

Registered

Unknown

Registered

Molecular weight

198

212

198

198

170

Physico-chemical properties

 

 

 

 

 

Appearance

Liquid

Liquid

Liquid

Liquid

liquid

Exp. Log Kow (Episuite)

4.7 (4.42)

5.4 (4.9)

(4.46)

(4.42)

(3.55)

Structural alerts for sensitisation

 

 

 

 

 

OECD Toolbox

No

No

No

No

No

Human toxicological information

 

 

 

 

 

Skin sensitisation animal tests

100%-negative

OECD 406, Kl 2

EC3>100%

(LLNA, OECD TG 429), Kl14.9)

EC3 > 25% (maximum tolerable dose)

 

20%-negative

OECD TG 406, Kl 2

No data

Skin sensitisation

HRIPT*^

10%-negative

Api, 2002

101 subjects

Kl 2

No relevant data

100%-negative

Api, 2002

52 subjects

Kl 2

100%-negative

Api, 2002

52 subjects

Kl 2

100%-negative

Api, 2002

52 subjects

Kl 2

*Api, A.M., 2002, Sensitization methodology and primary prevention of the Research Institute for Fragrance Materials. Dermatology, 205, 84-87.

^ Politano, V.T. and Api, A.M., 2008, The Research Institute for Fragrance Materials' human repeated insult patch test protocol. Regul Toxicol Pharmacol., 52, 35-38.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The substance is not a respiratory sensitiser because there are no human data that indicate respiratory sensitisation. In addition the potential forrespiratory sensitization of the substance is assessed using the integrated evaluation strategy for respiratory sensitization data in the ECHA guidance (R7A, Fig. 7.3-2).

1)           The substance is not a strong skin sensitizer;

2)           The substance does not belong to the di-isocyanates;

3)           The substance has not structural alerts or is structurally related to chemicals causing respiratory sensitization as presented in Table R.7.3-1 in the ECHA guidance of 2008 or those provided in the following document: http://ec.europa.eu/health/scientific_committees/docs/annex6_respiratory.pdf

Using this ITS in the ECHA guidance it can be concluded that the substance is not a respiratory sensitiser.

Justification for classification or non-classification

Based on the available information test substance does not have to be classified as a skin or respiratory sensitiser in accordance with EU CLP (EC No. 1272/2008 and its updates).