2-(3,3-dimethylcyclohex-1-enyl)-2,5,5-trimethyl-1,3-dioxane

Endpoint summary

Description of key information

In chemico - DPRA: Positive (OECD 442C, GLP, WoE, Rel.1)

In vivo - LLNA: BrdU: Skin sensitiser (OECD 422-B, GLP, WoE, Rel. 1)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 October 2020 - 12 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 442C and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted on 04 February 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

Certificate for GLP compliance dated on 1st of October 2019.

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS

- Preparation of the peptide stock solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

- Preparation of Peptide Calibration Standards: Calibration standards of both peptides were prepared by serial dilution of the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534
mM. A buffer blank was also prepared.

- Preparation of the test chemical solutions: the test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetonitrile) at 100 mM. This formulation was colorless limpid solution and was used just after its preparation.
For the reactivity of test item with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of test item with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

- Preparation of the positive controls, reference controls and co-elution controls:
* Precision controls (Reference Control A, n=3) and stability controls (Reference Control B, n=6), of both peptides were prepared at a peptide concentration of 0.5 mM. Reference Control A was used to assess the precision of injection, while Reference Control B was used to verify the stability of the peptide response over time and as a comparison point to determine depletion of the peptide in test samples.
* The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile.
* Accurate volume aliquots of the positive control were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of the positive control. For the co-elution controls, buffer solution was used in place of the Cysteine stock solution.
* Accurate volume aliquots of the positive control were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of the positive control. For the co-elution controls, buffer solution was used in place of the Lysine stock solution.

INCUBATION:

Incubation conditions: the appearance of the test substance and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run.
Before initiation of the run the appearance of the samples in the vials was assessed visually to check the absence of precipitate and documented again.

PREPARATION OF THE HPLC

- Instrumentation Parameters:
The HPLC/UV method used for the samples analysis according to the recommendations of the OECD guideline No. 442C is described in Covance internal analytical method and is summarized in the table below:
* HPLC: Waters Alliance 2695 separation module and 2487 dual wavelength detector
* Analytical Column: Agilent Zorbax SB C18, 100 x 2.1 mm, 3.5 μm
* Guard column: Phenomenex AJO4286
* Column temperature: 30°C
* Sample temperature: 25°C
* Mobil phase: Mobile phase A: 0.1% TFA in water / Mobile phase B: 0.085% TFA in Acetonitrile.
* Gradient:
Time % Mobile phase A % Mobile phase B
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
* Flow rate: 350 µL/minute
* Strocke volume: 25 µL
* Detector wavelength: UV, 220 nm
* Injection volume: 2 µL
* Retention times: Cysteine-peptide: approx. 11 minutes / Lysine-peptide: approx. 6.8 minutes
* Total analysis time: 30 minutes

- Verification of the suitability of the HPLC for test chemical and control substances: yes.
* Monitor retention time and tailing factor for each analytical run.
* Retention time and tailing factor should not deviate beyond the limits set below for the highest calibration standard (standard 6 of each peptide).
* Limits for system suitability: lysine peptide:
Retention time (min) +/- 10%: 6.84 (Nominal), 6.16 (Minimum), 7.52 (Maximum)
Tailing factor +/- 10%: 1.50 (Nominal), 1.35 (Minimum), 1.65 (Maximum)

* Limits for system suitability: Cysteine peptide:
Retention time (min) +/- 10%: 11.26 (Nominal), 10.13 (Minimum), 12.39 (Maximum)
Tailing factor +/- 10%: 1.43 (Nominal), 1.29 (Minimum), 1.57 (Maximum)

DATA EVALUATION:

- Cys and Lys peptide detection wavelength: The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - [Peptide peak area in replicate depletion samples (x 100)/Mean Peptide peak area of reference control samples B or C].

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Cinnamic aldehyde was used as a positive control.
- Result of positive control samples in cysteine assay: Mean % depletion = 70.6% and SD = 0.17%
- Result of positive control samples in lysine assay: Mean % depletion = 57.8% and SD = 0.61%
Mean depletion rate of Cinnamaldehyde: 64.2%
The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).

Key result

Parameter:

other: Mean % depletion in Cysteine assay

Value:

93.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Reactivity Class: High reactivity

Remarks:

DPRA Predicition: Positive.

Key result

Parameter:

other: Mean % depletion in Lysine assay

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Reactivity Class: No or minimal reactivity

Remarks:

DPRA Prediction: Negative.

Key result

Parameter:

other:

Remarks:

Mean depletion rate

Value:

46.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Reactivity Class: High reactivity

Remarks:

DPRA Prediction: positive

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
Observation of assay samples for both Cysteine and Lysine peptides indicated that the test item was not fully soluble in the assay media.
In Lysine assay samples and the co-elution control, there was evidence of phase separation, with a distinct “lens” of separated material noted on the solution surface. The dissolution control (test item in acetonitrile) was a clear colourless solution.
In Cysteine assay samples, a cloudy appearance was noted, attributed to the presence of a fine white precipitate. In contrast, the dissolution control was a clear colourless solution, while the co-elution control showed evidence of phase separation similar to the Lysine assay samples.
The precipitate present in Cysteine assay samples may be attributed to reaction products of the peptide and the test substance.

There were no co-elution peaks in either the Cysteine or Lysine assays.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: not specified.

Solubility results

The solubility of the test item in acetonitrile at a nominal concentration of 100 mM was achieved.
Observation of assay samples for both Cysteine and Lysine peptides indicated that the test item was not fully soluble in the assay media. 

Reactivity Assessment: 
The DPRA prediction and the reactivity of the test substance based on the overall mean and the individual depletion values in the Cysteine peptide and the Lysine peptide is presented in Table 7.4.1/1 and Table 7.4.1/2. 

 

Interpretation of results:

other: Positive in the DPRA assay.

Conclusions:

Under the experimental conditions of this study, the mean depletion of both peptides was 46.9%. Therefore, the test item was considered to have high peptide reactivity and hence it is predicted by DPRA to be positive in terms of being a potential skin sensitizer.

Executive summary:

The skin sensitisation potential of the test item was evaluated using an in chemico direct peptide binding assay using the validated DPRA method (Covance analytical method FIA/M101/15, based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) and in compliance with GLP.

 

 The reactivity of the test item was evaluated by monitoring peptide depletion following an approximate 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for a minimum of 22 hours in glass autosampler vials, protected from light and set at 25°C. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm.

The test item was dissolved at 100 mM in acetonitrile without sonication.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

The Observation of assay samples for both Cysteine and Lysine peptides indicated that the test item was not fully soluble in the assay media.

In Lysine assay samples and the co-elution control, there was evidence of phase separation, with a distinct “lens” of separated material noted on the solution surface. The dissolution control (test item in acetonitrile) was a clear colourless solution.

In Cysteine assay samples, a cloudy appearance was noted, attributed to the presence of a fine white precipitate. In contrast, the dissolution control was a clear colourless solution, while the co-elution control showed evidence of phase separation similar to the Lysine assay samples. The precipitate present in Cysteine assay samples may be attributed to reaction products of the peptide and the test substance.

As described in the OECD Guideline 442C, if a precipitate is observed immediately upon addition of the test chemical solution, due to low aqueous solubility of the test chemical, although one cannot be sure how much test chemical remained in the solution to react with the peptide, in such case, a positive result can still be used. Therefore, the cysteine depletion values is considered as valid.

The cysteine depletion value was 93.4%, the lysine depletion value was 0.3% and the mean of the cysteine and lysine depletion values was 46.9%. All acceptance criteria were met.

 

Under the experimental conditions of this study, the test item was considered to have high peptide reactivity and hence it is predicted by DPRA to be positive in terms of being a potential skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Between 22 July to 11 August, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 442-B.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA or –FCM)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.51 (Skin Sensitisation: Local Lymph Node Assay: BrDU-ELISA)

Version / remarks:

30 May 2012

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

13 September 2013.

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Age at study initiation: 8 weeks
- Weight at study initiation: 18.9 - 21.2 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: food (A04, SAFE), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: At least 10 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From 22 July to 11 August 2015.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary screening test: 100%
Main test: 25% (v/v), 50% (v/v) and 100% (w/w) in the vehicle Acetone/olive oil (4/1, v/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- As no available information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at 100% to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on day 1, day 3 and on day 6. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.
- Irritation: No mortality and no signs of systemic toxicity were noted. Slight dryness was noted on day 6 at the concentration of 100%. No other cutaneous reactions were noted at the concentration of 100%.
Therefore, the concentration of 100% was chosen as the highest concentration for the main study.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA: BrdU)
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1. 6 compared to control values. Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI < 1.6 will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
25 μL of control or test substance was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised. From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a 70 nylon mesh in 15 mL of PBS (Ca2+/Mg2+-free) into a well of multi-well 6. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1-0.2.

Stimulation index determination:
BrdU was measured by ELISA using a commercial kit (e;g; Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001-Batch No.11417600). Briefly, 100 µL of the LNC suspension was added to the wells of a flat-bottom microplate at least in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substracte solution was then added and allowed to produce chromogen. After 5 to 30 min, 25 Ul of 1 M H2SO4 was added in each well, then shaked for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured. The BrdU labelling index was defined as: BrdU labelling index = (ABSem-ABS blankem) - (ABS ref-ABS blank ref).
em: emission wavelength; and ref = reference wavelength.

Positive control substance(s):

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

Statistics:

None.

Positive control results:

One group of four animals, were treated with 50 μL (25 μL per ear) of α- Hexylcinnamaldehyde, as a solution in acetone/olive oil (4:1, v/v) at concentration 25% (v/v). With EC1.6 = 2.32, α-Hexylcinnamaldehyde is considered to be a sensitiser under the conditions of the test.

Key result

Parameter:

SI

Value:

1.33

Test group / Remarks:

25% (v/v) in Acetone/Olive oil (4:1)

Key result

Parameter:

SI

Value:

1.46

Test group / Remarks:

50% (v/v) in Acetone/Olive oil (4:1)

Key result

Parameter:

SI

Value:

1.61

Test group / Remarks:

100% (v/v) in Acetone/Olive oil (4:1)

Key result

Parameter:

other: EC1.6

Remarks:

%

Value:

96.67

Test group / Remarks:

Test item

Cellular proliferation data / Observations:

- A stimulation index of more than 1.6 was recorded for one concentration of the test item (100% (v/v) in acetone/olive oil).
The Stimulation Index (SI) calculated by pooled approach was respectively 1.33, 1.46 and 1.61 for the treated group at 25%, 50% and 100%. The EC1.6 value determined by linear interpolation of points on the dose-response curve was 96.67%.

LOCAL IRRITATION
- No cutaneous reaction was noted in the treated animals.
- No significant increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100%. Therefore, the test item must be considered as not excessively irritant at the three concentrations.

CLINICAL OBSERVATIONS
- No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.

BODY WEIGHTS
- Bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the same period.

None.

Interpretation of results:

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

Conclusions:

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, three groups of CBA/J (CBA/J@Rj) strain mice (4 females/dose) were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item at concentrations of 25%, 50% and 100% (v/v) in Acetone/Olive oil (4:1). A further group of four animals was treated with Acetone/Olive oil (4:1). At D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The test concentrations for the main study were determined from a preliminary study tested at 100 %. Based on the results of preliminary study, the dose levels selected for the main test were 25%, 50% and 100% (v/v) in Acetone/Olive oil (4:1).

 

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. No significant increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100%. Therefore, the test item must be considered as not excessively irritant at the three concentrations.

A stimulation index of more than 1.6 was recorded for one concentration of the test item (100% (v/v) in acetone/olive oil). The Stimulation Index (SI) calculated by pooled approach was respectively 1.33, 1.46 and 1.61 for the treated group at 25%, 50% and 100%. The EC1.6 value determined by linear interpolation of points on the dose-response curve was 96.67%.

 

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation (EC) No. 1272 /2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin sensitisation endpoint.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Two studies are available to assess the skin sensitization potential of the substance.

1/ A local lymph node assay was performed according to OECD Guideline 422-B and in compliance with GLP.

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. No significant increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100%. Therefore, the test item must be considered as not excessively irritant at the three concentrations.

A stimulation index of more than 1.6 was recorded for one concentration of the test item (100% (v/v) in acetone/olive oil). The Stimulation Index (SI) calculated by pooled approach was respectively 1.33, 1.46 and 1.61 for the treated group at 25%, 50% and 100%. The EC1.6 value determined by linear interpolation of points on the dose-response curve was 96.67%.

Under these experimental conditions, the test material is a skin sensitizer.

2/ A Direct Peptide Reactivity Assay was performed according to OECD Guideline 442C and in compliance with GLP.

The cysteine depletion value was 93.4%, the lysine depletion value was 0.3% and the mean of the cysteine and lysine depletion values was 46.9%. All acceptance criteria were met. The test material was considered to be positive in the Direct Peptide Reactivity Assay and was considered to have high reactivity.

 

Therefore, the substance is considered to be a skin sensitizer under both the in chemico and in vivo assays.

 

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self-classification:

Based on the available data, the substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the CLP and to the GHS as the LLNA EC1.6 is > 6%.

 

No data was available regarding respiratory sensitisation.