2,4,6-trimethylcyclohex-3-enecarbaldehyde

Endpoint summary

Description of key information

LLNA (OECD 429): Stimulation index>3. Isocyclocitral in 1:3 EtOH:DEP vehicle is a skin sensitiser under the conditions of the test with an EC3 value of 7.3% (1825µg/cm²).

KeratinoSens Assay (in vitro): In all three repetitions, no induction of the luciferase above the threshold of 1.5 was noted. According to the prediction model of the KeratinoSens™assay, ISOCYCLOCITRAL TECH is rated as non-sensitizer. This is also clearly supported by the analysis of the dose-response curve in Figure 4 with overall no induction of the luciferase reporter gene to be observed.

DPRA Prediction Model: ISOCYCLOCITRAL TECH was reactive and classified into the MODERATE reactivity class according to the prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was conducted between 2 November 2005 and 20 December 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The LLNA study was performed in 2006 and is used as a key study in this dossier. A two in-vitro studies (DPRA and Keratinosense) were performed later-on in 2015 and also included into this dossier as additional useful information regarding correlation between in-vitro and in-vivo data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca/Ola/Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: Young adults (8-12 weeks of age)
- Weight at study initiation: 15-21 g
- Housing: Animals were group housed (4 per cage)
- Diet: RM1, supplied by Special Diet Services Limited, Witham, Essex, UK ad libitum
- Water: Mains water was available ad libitum
- Acclimation period: Study animals were acclimated to their housing for at least five days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/-3 °C
- Humidity: 30-70 %
- Air changes (per hr): 15 minimum
- Light cycle: Atrificial, giving 12 hrs light, 12 hrs dark
- Environmental enrichment included tents, bases and nestlets.

Vehicle:

other: Diethyl phthalate/ethanol in a ratio of 3:1

Concentration:

0.5%, 1.0%, 2.5%, 5% and 10% (test article)
5%, 10% and 25% (positive control) in acetone in olive oil (4:1).

No. of animals per dose:

4

Details on study design:

Groups of four female mice were used for this study. Approximately 25µl of a 0.5, 1, 2.5, 5 or 10% w/v preparation of the test substance in 1:3 EtOH:DEP was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using 1:3 EtOH:DEP alone. The procedure was repeated daily for 3 consecutive days.

Three days after the third application, all the animals were injected, via the tail vein, with approximately 250µl of phosphate buffered saline (PBS) containing approximately 20µCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halthane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10ml of PBS. Approximately 3ml of 5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatent was discarded. The cells were then resuspended in approximately 1ml of TCA.

The lymph node suspensions were transferred to scintillation vials and 10ml of scintillant (Optiphase) was added prior to ß-scintillation counting using a Packard Tr-Carb 3100TR Liquid Scintillation Counter.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The results are expressed as a disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test:control ratio known as the stimulation index (SI), for each concentration. The criterion for a positive response is that one or more concentrations of a test article should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. The assay is able to identify those materials that elicit responses in standard guinea pig tests for skin sensitisation (Kimber et al 1994). Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a skin sensitiser.

Positive control results:

The application of hexylcinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone in olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at the 10 and 25% w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

Parameter:

SI

Value:

0.8

Test group / Remarks:

Concentration of test substance [%w/v]: 0.5

Parameter:

SI

Value:

1.1

Test group / Remarks:

Concentration of test substance [%w/v]: 1

Parameter:

SI

Value:

1.7

Test group / Remarks:

Concentration of test substance [%w/v]: 2.5

Parameter:

SI

Value:

1.8

Test group / Remarks:

Concentration of test substance [%w/v]: 5

Parameter:

SI

Value:

4.4

Test group / Remarks:

Concentration of test substance [%w/v]: 10

Parameter:

EC3

Value:

7.3

Table 1 Skin Sensitisation Potential of Isocyclocitral

 

* Diethyl phthalate in a ratio of 3:1

Test/control ratio of 3.0 or greater represents a positive result

N/A - not applicable

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Isocyclocitral in 1:3 EtOH:DEP vehicle is a skin sensitiser under the conditions of the test with an EC3 value of 7.3% (1825µg/cm2).

Executive summary:

Study design

A sample of Isocyclocitral was assessed for its skin sensitisation potential using the mouse Local Lymph Nodc Assay. The assay determines the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application, by measuring the amount of radiolabelled thymidine incorporated into dividing cells. The test substance was applied as 0.5, 1, 2.5, 5 or 10% w/v preparations in 1:3 Ethanol/Diethylphtalate (1:3 EtOH:DEP, supplied by the sponsor). A vehicle control group was similarly treated using 1:3 EtOH:DEP alone. Dose levels were set by the sponsor in accordance with the acute oral toxicity data provided. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated as percentage dose and µg/cm².

Results

The test substance had the capacity to cause skin sensitisation when applied as a dose of 10% w/v preparation in 1:3 EtOH:DEP. The EC3 value giving rise to a 3 fold increase in lymphocyte proliferation was calculated to be 7.3% w/v (1825 µg/cm²).

 In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 10 and 25 % w/v preparations in acetone in olive oil (4:1), confirming the validity of the protocol used for ths study.

Conclusion

In conclusion, Isocyclocitral in 1:3 EtOH:DEP vehicle is a skin sensiiser under the conditions of the test with an EC3 value of 7.3% (1825 µg/cm²).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 26, 2015 to February 5, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The assay underwent validation at the European Centre for the Validation of Alternative Methods (ECVAM) [9] and an OECD test guideline was adopted (OECD TG 442d). The protocol was published as DB-ALM protocol 155 [1]. The assay was proposed by ECVAM [9] to be to be used as part of an integrated approach for testing and assessment (IATA)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

Principles of method if other than guideline:

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response.
This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to skin sensitizers from a broad range of so called applicability domains, ie. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA)

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The Cosmetic directive is phasing out of animal testing for new products. This has led to the
development of potential alternative assays to screen for sensitizing potential. These new assays
were tested against a large list of reference chemicals in order to prove their applicability domain.
The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin
sensitizers by their ability to induce the Nrf2-response

Details on the study design:

Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a final level of 1% of the solvent DMSO containing the test substance. Each compound is tested at 12 concentrations in the range from 0.98 to 2000 µM. Each test plate contains six wells with the solvent control, 1 well with no cells for background value and 5 wells with a dose response of the positive control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same set-up, and a forth parallel plate is prepared for cytotoxicity determination.

Sex:

not specified

Details on test animals and environmental conditions:

Not applicable.

No. of animals per dose:

Not applicable.

Positive control results:

Cinnamic aldehyde was run in all three repetitions. Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in all three repetitions. The induction at 64 µM and the EC 1.5 for cinnamic aldehyde were also calculated. The targets are: (i) Average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8, and (ii) the EC 1.5 value should be between 7 µM and 30 µM. At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed here both criteria were fulfilled in all three repetitions. Thus all three repetitions were valid for the positive control.
As second performance criterion, the variability of the solvent control must be below 20%. All three repetitions were valid for the solvent control.

Run / experiment:

other: 1

Parameter:

other: Average of Imax (fold induction)

Value:

1.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Cytotoxicity determinations.Given is the IC50 value as the concentration inµM reducing the viability by 50%.

Luciferase determinations.Given is the I_maxvalues indicating maximal fold-induction up to a concentration of 1000µM.

 

Overall rating of the test substance according to the prediction model and the number of positive repetitions.

Interpretation of results:

study cannot be used for classification

Remarks:

Criteria used for interpretation of results: other: ECVAM, KeratinoSensTM protocol. DB-ALM database, 2014. Protocol 155.

Conclusions:

In all three repetitions, no induction of the luciferase above the threshold of 1.5 was noted. According to the prediction model of the KeratinoSens™ assay, the test substance is rated as non-sensitizer. This is also clearly supported by the analysis of the dose-response curve in Figure 4 with overall no induction of the luciferase reporter gene to be observed.

Executive summary:

Introduction

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin

sensitizers by their ability to induce the Nrf2-response.

This assay has been validated for a broad range of low-molecular weight chemicals and it was found

to respond to skin sensitizers from a broad range of so called applicability domains, i.e. chemicals

reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be

used as part of an integrated approach for testing and assessment (IATA).

Experimental

The test substance ISOCYCLOCITRAL TECH was dissolved in DMSO and tested according to the

standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions,

each time in three replicates. After 48 h incubation time, luciferase induction and cellular viability at

each of the concentrations were determined.

Results

ISOCYCLOCITRAL TECH had low toxicity to the KeratinoSens™ cells with an IC50 of 596 μM. It did

not induce the luciferase gene above a threshold of 1.5 in all three repetitions. It is therefore

considered a non-sensitizer according to the prediction model of the KeratinoSens™ assay.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

January 26, 2015 to January 30, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The Cosmetic directive is phasing out of animal testing for new products. This has led to the
development of potential alternative assays to screen for sensitizing potential. These new assays
were tested against a large list of reference chemicals in order to prove their applicability domain.
The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test
substances towards peptides.

Details on the study design:

The Lys-peptide Ac-RFAAKAA is incubated at a final concentration of 0.5 mM in an ammonium
acetate buffer at pH 10.5 in presence of a final level of 25% acetonitrile and in presence of a 50-fold
excess of the test substance (25 mM) dissolved in acetonitrile.
The Cys-peptide Ac-RFAACAA is incubated at a final concentration of 0.5 mM in phosphate buffer
at pH 7.5 in presence of a final level of 25% acetonitrile and in presence of a 10-fold excess of the
test substance (5 mM) dissolved in acetonitrile.

24 h after start of the incubation the remaining peptide is quantified with HPLC-UV.

Sex:

not specified

Details on test animals and environmental conditions:

Not applicable.

No. of animals per dose:

Not applicable

Run / experiment:

other: Cys-peptide depletion

Value:

23

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Lys-peptide depletion

Value:

51

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:


- Acceptance criteria met for positive control:
Cys-peptide depletion: 60.8 – 100%
Lys-peptide depletion: 40.2- 69.4%
- Acceptance criteria met for variability between replicate measurements:
Cys-peptide depletion: coefficient of variation < 14.9
Lys-peptide depletion: coefficient of variation < 11.6

Average Standard deviation Cys-peptide depletion 25.5 2.2 Lys-peptide depletion 52.0 0.2 Average depletion Cys-and Lys-peptide 38.7  Reactivity Class MODERATE  Prediction Sensitizer  Elution time Cys peptide 10.7  Elution time test substance Cys peptide run 15 / 15.8 (2 Peaks)  Elution time Lys peptide 8.12  Elution time test substance Lys peptide run 15 / 15.8 (2 Peaks) 

Interpretation of results:

study cannot be used for classification

Remarks:

Key Study is used for classification

Conclusions:

ISOCYCLOCITRAL TECH was reactive and classified into the MODERATE reactivity class according to the prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.

Executive summary:

ISOCYCLOCITRAL TECH was reactive and classified into the MODERATE reactivity class according to the prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

The results from two in-vitro assays gave conflicting results:

1. The KeratinoSens Assay gave a negative result.

2. The DPRA prediction model suggested moderate activity and that the substance be classed as a sensitiser.

The LLNA (OECD 429) study by Betts performed in 2005/6 indicated Isocyclocitral in 1:3 EtOH:DEP vehicle to be a skin sensitiser under the conditions of the test with an EC3 value of 7.3% (1825µg/cm²). Thus the overriding evidence is that the substance be classified as a skin sensitizer according to the test results. The LLNA study combinated with DPRA study are relevant and conclude classification of the substance.