1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 429, EPA OPPTS 870.2600, EC Method B.42 and in accordance with the Principles of Good Laboratory Practices (GLP)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

EC Number:

600-447-7

Cas Number:

14228-73-0

Molecular formula:

C14 H24 O4

IUPAC Name:

1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Details on test material:

- Name of test material (as cited in study report): 1,4 Cyclohexanedimethanol Reaction Products with Epichlorohydrin
- Analytical purity: The purity of the test material was determined to be 9.5 area % 1,4-Cyclohexanedimethanol monoglycidiyl ether, 45.0 area %, 1,4-Cyclohexanedimethanol diglycidyl ether, and 25.0 area % of the reaction product of 1,4-cyclohexanedimethanol with 3 equivalents of epichlorohydrin by gas chromatography/flame ionization detector with tentative identification of the major components by nuclear magnetic resonance and gas chromatography mass spectrometry
- Impurities (identity and concentrations): no information available
- Lot number : Lot # 1H1050NZP1
- Expiration date of the lot/batch: March 05, 2015

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan (Indianapolis, Indiana)
- Age at study initiation: 9-12 weeks
- Housing: Animals were housed up to six per cage in filter tubs containing corncob bedding, food pellets and a water bottle.
- Diet : ad libitum - Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form
- Water : ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0.5%, 2% and 10% dosing solutions were prepared daily just prior to dosing. The concentrations of the dosing solutions were not verified analytically.

No. of animals per dose:

6 mice/dose group

Details on study design:

RANGE FINDING TESTS:
A pre-screen test was performed in order to define the maximum dose level for use in the LLNA. The ears of six mice (one female/concentration) were topically treated for three consecutive days with one of six concentrations (0%, 5%, 10%, 25%, 50%, or 100%) of 1,4-CHDM DGE. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to dosing on Day 1 and prior to termination on Day 6. Ear thickness measurements were taken for each ear using a digital micrometer (Mitutoyo, Japan; Model 700-122) prior to dosing on Day 1 and 3 and on Day 6 prior to termination.
During the screening study, the mice were treated with three daily applications of 0%, 5%, 10%, 25%, 50%, or 100% of 1,4-CHDM DGE. Erythema was absent in the mouse treated with 5% 1,4-CHDM DGE, while the mice treated with 10%, and 25% demonstrated slight erythema on day 3 which resolved by day 6. The mouse dosed with 50% demonstrated well-defined erythema on day 3 which resolved by day 6. The mouse dosed with 100% 1,4-CHDM DGE demonstrated slight erythema on day 2, which progressed to well-defined by day 3 and regressed to slight erythema by day 6. Ear swelling increases of greater than 25% were observed for all mice treated with 1,4-CHDM DGE on day 3 and for mice treated with 25%, 50%, and 100% 1,4-CHDM DGE on day 6.
Based on the results of the screen, 10% 1,4-CHDM DGE was tested in the LLNA along with 2% and 0.5% to characterize the dose response. The 10% concentration was chosen as the top dose based on consideration of both erythema and ear swelling data from the irritation screen. Although ear swelling of greater than 25% was noted at the 10% concentration, as this was without well-defined erythema and did not persist through day 6. Therefore the 10% concentration was considered an appropriate high concentration for the main LLNA study.

MAIN STUDY
Dimethyl sulfoxide (DMSO) was selected based upon maximum miscibility of 1,4-CHDM DGE while maintaining a solution suitable for application. α-hexylcinnamaldehyde (HCA) solution was used as a positive control for contact sensitization. HCA (Aldrich Chemical, Milwaukee, Wisconsin, Cat. No. W256900) was diluted to 25% (v/v) in vehicle (DMSO). Analytical verification of the solution was not performed.

The application of the test material (25 μl/ear) was made on the dorsal surface of both ears. Five female mice/group received one of three concentrations of
1,4-CHDM DGE (0.5%, 2%, or 10%) or vehicle (DMSO) once daily for three consecutive days. HCA at 25% (v/v) in vehicle was run concurrently as a positive dermal sensitization control. Ears were inspected prior to application of the test material solutions, and erythema was evaluated on days 1, 2, 3, and 6. All mice were weighed on days 1 and 6. On day 6, all mice received a 250 μl intravenous injection (i.v.) via the lateral tail vein containing 20 μCi of 3H-thymidine (specific activity 2 Ci/mmol; Perkin Elmer product number NET027A001MC) diluted in phosphate-buffered saline (PBS). Approximately four to five hours post administration, the mice were euthanized via CO2 asphyxiation and both auricular lymph nodes located at the bifurcation of the jugular veins were excised and placed in PBS. A single cell suspension of the auricular lymph nodes from one mouse was prepared by gentle mechanical disaggregation using a tissue homogenizer (Stomacher 80 Lab System, Seward Ltd., London, United Kingdom). The cells were washed two times and were suspended in 3 ml of 5% trichloroacetic acid (TCA) for approximately 18-70 hours. The suspended precipitates were centrifuged (200 x g for 10 minutes) and the supernatant removed. The pellet from each mouse was reconstituted in 1 ml of 5% TCA and subsequently transferred to a scintillation vial containing 10 ml of Aquasol-2 scintillation cocktail (Packard Instrument Company, Meriden, Connecticut). Two additional 2 ml aliquots of water were used to rinse the tubes and the rinses were added to the scintillation vials containing the 1 ml of pellet in TCA and cocktail. The radioactivity in each precipitate was measured using a β-scintillation counter and reported as disintegrations per minute (dpm) per mouse. A mean dpm value + SD (standard deviation) was calculated for each experimental group. The SI was calculated using the absolute dpm value for each mouse as the numerator and the mean dpm value from the vehicle control mice as the denominator; the mean SI + SD was calculated for each experimental group.

Positive control substance(s):

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

Statistics:

Standard statistical methods were employed

Results and discussion

Positive control results:

Proper conduct of the LLNA was demonstrated via the positive response from the positive control, 25% HCA which elicited a stimulation index (SI) that was 6.7 in comparison to vehicle-treated mice.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

Highest test conc 10%

Value:

2.9

Test group / Remarks:

Si = 2.9 at 10% conc (highest test conc.)

Remarks on result:

other: see Remark

Remarks:

1,4-cyclohexanediemethanol, reation products with epichlorohydrin (1,4-CHDM DGE) at concentrations of 0.5%, 2% and 10% elicited proliferative responses with stimulation indices (SI) of 1.7, 2.3, and 2.9, respectively, in comparison to vehicle-treated mice.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The mean dpm's of various groups were as follows - Vehicle control - 880.4 0.5% - 1459.8 2% - 2021.8 10% - 2574.6 25% HCA - 5934.2

Results 3

Key result

Parameter:

SI

Remarks:

2% test conc.

Value:

2.3

Test group / Remarks:

2% test conc.

Results 4

Key result

Parameter:

SI

Remarks:

0.5% test conc

Value:

1.7

Test group / Remarks:

0.5% test conc

Any other information on results incl. tables

In the LLNA erythema was absent in the mice treated with 0.5% and 2%, while 4/5 mice dosed with 10% demonstrated slight erythema on day 3 and body weights were unaffected in all dose groups.

Applicant's summary and conclusion

Interpretation of results:

other: ambiguous

Remarks:

1,4-CHDM DGE belongs to a class of chemistries (diglycidyl ethers/epoxies) that have been well-established to have dermal sensitization potential. Therefore, at concentrations greater than 10%, 1,4-CHDM DGE may have weak dermal sensitization potential.

Conclusions:

1,4-cyclohexanediemethanol, reaction products with epichlorohydrin (1,4-CHDM DGE) at concentrations of 0.5%, 2%, and 10% elicited proliferative responses with stimulation indices (SI) of 1.7, 2.3, and 2.9, respectively, in comparison to vehicle-treated mice. Based on these results, 1,4-CHDM DGE did not demonstrate dermal sensitization potential in the mouse LLNA at concentrations up to and including 10%, as the lymph nodes draining the area of topical application did not demonstrate a 3-fold increase in proliferation when compared to vehicle-treated mice.
However, 1,4-CHDM DGE belongs to a class of chemistries (diglycidyl ethers/epoxies) that have been well-established to have dermal sensitization potential. Therefore, at concentrations greater than 10%, 1,4-CHDM DGE may have weak dermal sensitization potential

Executive summary:

The Local Lymph Node Assay (LLNA) was conducted to assess the potential of 1,4- Cyclohexanedimethanol, reaction products with epichlorohydrin (1,4-CHDM DGE) to cause contact sensitization by measuring lymphocyte proliferative responses from auricular lymph nodes following topical application of the test material to the mouse ear.

Screening Study: Three daily topical applications of 0%, 5%, 10%, 25%, 50%, or 100% 1,4-CHDM DGE were given to one animal at each dose level. Erythema was absent in the mouse treated with 5% 1,4-CHDM DGE, while the mice treated with 10%, and 25% demonstrated slight erythema on day 3 which resolved by day 6. The mouse dosed with 50% demonstrated well-defined erythema on day 3 which resolved by day 6. The mouse dosed with 100% 1,4-CHDM DGE demonstrated slight erythema on day 2, which progressed to well-defined by day 3 and regressed to slight erythema by day 6.. Ear swelling increases of greater than 25% were observed for all mice treated with 1,4-CHDM DGE on day 3 and for mice treated with 25%, 50%, and 100% 1,4-CHDM DGE on day 6. Body weights were unaffected in all dose groups. Results from this study were used to determine the dosing concentrations for 1,4-CHDM DGE in the LLNA.

LLNA: Five female mice/group received 0.5%, 2%, or 10% of 1,4-CHDM DGE, or vehicle (DMSO) or 25% α-hexylcinnamaldehyde (HCA; positive control) on days 1-3. On day 6, uptake of 3H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 25% α-hexylcinnamaldehyde (HCA), a moderate contact sensitizer, which elicited proliferation with a stimulation index of 6.7 in comparison to vehicle-treated mice.

Erythema of the ears was absent in the mice treated with 0.5% and 2% 1,4-CHDM DGE, while 4/5 of the mice treated with 10% demonstrated slight erythema on day 3, which was resolved by day 6. Body weights were unaffected in all dose groups.

1,4-cyclohexanediemethanol, reaction products with epichlorohydrin (1,4-CHDM DGE) at concentrations of 0.5%, 2%, and 10% elicited proliferative responses with stimulation indices (SI) of 1.7, 2.3, and 2.9, respectively, in comparison to vehicle-treated mice.

Based on these results, 1,4-CHDM DGE did not demonstrate dermal sensitization potential in the mouse LLNA at concentrations up to and including 10%, as the lymph nodes draining the area of topical application did not demonstrate a 3-fold increase in proliferation when compared to vehicle-treated mice. However, 1,4-CHDM DGE belongs to a class of chemistries (diglycidyl ethers/epoxies) that have been well-established to have dermal sensitization potential. Therefore, at concentrations greater than 10%, 1,4-CHDM DGE may have weak dermal sensitization potential.