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Description of key information

As no conclusion can be drawn on the skin sensitizing potential of the test item based on the in vitro and in chemico data-set, and cell-based assays do not give reliable results for this substance to further determine this endpoint, it is considered scientifically justified to address the skin sensitizing properties of 1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) with an in vivo test.

 

The LLNA results indicate that the test item could elicit a SI≥3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 6.8% was calculated. According to the CLP, the test item should be classified as s skin sensitizer (Category 1B).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June to 20 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSens assay, which is recommended in international guidelines (e.g. OECD 442D).
Specific details on test material used for the study:
Appearance: Clear colourless liquid
Batch: 170918339
Purity: 97.2%
Details on study design:
Plating of cells:
- For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

Treatment of cells:
- The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0 °C in the presence of 5% CO2. In total 3 experiments were performed.

Luciferase Activity Measurement:
- The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment:
- For the KeratinoSens cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite M200 Pro Plate Reader.

Positive control results:
The EC1.5 of the positive control was between 5 and 125 µM (49 µM, 77 µM and 83 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.41-fold, 2.12-fold and 2.68-fold in experiment 1, 2 and 3, respectively).
Parameter:
other: EC1.5 (µM)
Run / experiment:
Experiment 1
Value:
2.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
Parameter:
other: EC 1.5 (µM)
Run / experiment:
Experiment 2
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.43 and therefore no EC1.5 could be calculated.
Parameter:
other: EC1.5 (µM)
Run / experiment:
Experiment 3
Value:
5.9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 1.71 and the EC1.5 5.9 µM.
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Three independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.004 – 7.8 µM (2-fold dilution steps) in experiment 1 and of 0.090 – 7.8 µM (1.5-fold dilution steps) in experiments 2 and 3 for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

One deviation was reported:

One of the luminescence readings for the DMSO vehicle control (9050) was excluded from the analysis of the positive control in experiment 1 as an outlier, since the variation was with 22.4% > 20%. And one of the luminescence readings for the EtOH vehicle control (2435) was excluded from the analysis of the test item in experiment 3, since the variation was with 20.2% > 20%. Based on evaluation of the data with Dixon’s Q-test these values were clear outliers.
Evaluation: After excluding this reading, still 17 vehicles were left for the calculations with a variation of 11% in experiment 1 and 3. Before excluding all other acceptation criteria were met. The exclusion was only affecting the positive control reading in experiment 1, which already met the acceptation criteria before exclusion. And in the third experiment had no influence on the positive result observed with the test item, since this was observed both before and after exclusion. Excluding these values has therefore no effect on the validity of the study.

This deviation was considered to not have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Interpretation of results:
study cannot be used for classification
Conclusions:
The test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in the study.  However, due to coinciding increases in luciferase induction and cell viability the biological relevance can be questioned.
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 May to 01 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
GLP compliance:
yes
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
Appearance: Clear colourless liquid
Batch: 170918339
Purity: 97.2%
Details on study design:
Solubility Assessent of the Test Item:
- At a concentration of 100 mM, 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) was not soluble in ACN, MQ, ACN:MQ (1:1, v/v), acetone:ACN (1:1, v/v) and DMSO:ACN (1:9, v/v), but was soluble in isopropanol. Therefore this solvent was used to dissolve the test item in this DPRA study.

Preparation of Solutions for Cysteine Reactivity Assay
- A stock solution of 0.667 mM Synthetic Peptide Containing Cysteine (SPCC) Stock Solution (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN. In addition, a RCcysCisopropanol sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCisopropanol sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL isopropanol. A SPCC calibration curve was prepared.
- The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared.

Preparation of Solutions for Lysine Reactivity Assay
- A stock solution of 0.667 mM Synthetic Peptide Containing Lysine (SPCL) Stock Solution (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN. In addition, a RClysCisopropanol sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCisopropanol sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL isopropanol. A SPCL peptide calibration curve was prepared.
- The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared.

Sample Incubations:
- After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature.

Data Evaluation:
- After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm.
Positive control results:
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 69.2% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
Parameter:
other: Percent Peptide Depletion
Run / experiment:
Cysteine Reactivity Assay (Cysteine 1:10)
Value:
0
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Percent Peptide Depletion
Run / experiment:
Lysine Reactivity Assay (1:50)
Value:
3.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Mean Percent Peptide Depletion
Run / experiment:
Mean of SPCC and SPCL depletion
Value:
1.9
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Test item was negative in the DPRA and was classified in the “no or minimal reactivity class”
Other effects / acceptance of results:
Precipitation was observed after the incubation period for both SPCC and SPCL. Therefore, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the negative result of this study is uncertain and should be interpreted with due care.
Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, this DPRA test met all acceptability criteria and is considered valid. The results suggest that the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 October 2018 to 16 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Appearance: Clear colourless liquid
Batch: 170918339
Purity: 97.2%
Species:
mouse
Strain:
CBA:J
Remarks:
Inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: ~11 weeks old
- Weight at study initiation: 20.6 to 23.7 g
- Housing: group housed (up to 5 animals of the same sex and same dosing group together)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 23°C
- Humidity (%): 42 to 52%.
- Air changes: Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod: 12 hrs dark / 12 hrs light
Vehicle:
methyl ethyl ketone
Remarks:
The vehicle was selected on the basis of maximizing the solubility based on trial preparations
Concentration:
Pre-screen Test: Initially, two test item concentrations were tested; a 50% and 100% concentration. Based on the results, two additional concentrations of 10% and 25% were tested.
Main Test: 2, 5, 10%
No. of animals per dose:
5/dose
Details on study design:
PRE-SCREEN TESTS:
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: the draining (auricular) lymph nodes were excised and pooled for each animal
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
In the main study, mice were treated with test item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone. Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
In case of borderline results, statistical analysis may be performed to determine the dose response relationship and pair wise comparisons between dose groups versus negative control.
Positive control results:
The results of a reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
Key result
Parameter:
EC3
Value:
ca. 6.8
Variability:
None
Key result
Parameter:
SI
Value:
ca. 1.5
Variability:
± 0.4
Test group / Remarks:
2%
Key result
Parameter:
SI
Value:
ca. 1.7
Variability:
± 0.1
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
ca. 5.4
Variability:
± 1.1
Test group / Remarks:
10%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA : The majority of auricular lymph nodes were considered normal in size, except for the nodes in four animals treated at 5% and all animals treated at 10%, which were considered enlarged.
The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION
The DPM value for one vehicle control animal was rejected and not used for interpretation. This value was outside the range normally seen for this vehicle control and was determined to be a significant outlier based on the Dixon's Q-test. For one animal dosed at 5%, the DPM value was rejected and not used for interpretation, as this value was determined to be a significant outlier based on the Dixon's Q-test. Sufficient data remained to warrant the study integrity.
Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 530, 607 and 1971 DPM, respectively. The mean DPM/animal value for the vehicle control group was 365 DPM.

CLINICAL OBSERVATIONS: The very slight erythema of the ears as shown by several animals treated at 5% and 10% between Days 1 and 5 was considered not to have a toxicologically significant effect on the activity of the nodes. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), the test substance should be classified as skin sensitizer (Category 1B). According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), the test substance should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.
Executive summary:

In a local lymph node assay, the test substance was tested to determine whether it induces skin sensitization in female CBA/J mice after three epidermal exposures of the animals. The study was conducted based on the OECD Test Guideline No. 429 under GLP conditions. Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 100%, 50% and 25% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore, these concentrations did not meet the selection criteria. At a 10% test item concentration, no signs of systemic toxicity were noted and very slight irritation only was observed and therefore this concentration was selected as highest concentration for the main study.

In the main study, mice (5/dose) were treated with test item concentrations of 0, 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (methylethylketone). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in four animals treated at 5% and all animals treated at 10%, which were considered enlarged. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 530, 607 and 1971 DPM, respectively. The mean DPM/animal value for the vehicle control group was 365 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 1.5, 1.7 and 5.4, respectively.

The results indicate that the test item could elicit a SI3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 6.8% was calculated. The six-month reliability check with alpha-hexylcinnamaldehyde indicates that the study is an appropriate model for testing for contact hypersensitivity. 

The test substance should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In the KeratinoSens assay, 1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) was tested positive. However, it is noted that the cells in this assay showed an a-typical response: a dose-related increase of viability instead of decrease was observed and the luciferase induction was almost superimposable with the cell vialibility. Based on this observation it was concluded that cell-based systems are not a reliable test system for this substance and hence further in vitro studies were omitted. Based on the results of a KeratinoSens assay that was conducted according to OECD 442D, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes.

 

In the DPRA study, precipitation was observed after the incubation period for both SPCC and SPCL. Consequently, it is uncertain how much test item remained in the solution to react with the peptides. Based on the results of a Direct Peptide Reactivity Assay (DPRA) study that was conducted according to OECD 442C, 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.  However, this negative result is uncertain and should be interpreted with due care.

As no conclusion can be drawn on the skin sensitizing potential of the test item based on the in vitro and in chemico data-set, and cell-based assays do not give reliable results for this substance to further determine this endpoint, it is considered scientifically justified to address the skin sensitizing properties of 1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) with an in vivo test.

In the LLNA study, the test item elicited as SI ≥ 3. The data showed a dose-response and an EC3 values of 6.8% was calculated.

Justification for classification or non-classification

In the LLNA study, the test item elicited as SI ≥ 3. The data showed a dose-response and an EC3 values of 6.8% was calculated. According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), 1,1’-(1,1,3-trimethylpropane-1,3-

diyl)bis(cyclohexane) should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.