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Description of key information

Two GLP and OECD giuideline studies and one non-guideline study were conducted for invetigating  the skin and eye irritation/corrosion effects of CHDM DGE . 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
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 439 and in accordance with the Principles of Good Laboratory Practices (GLP).
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Remarks:
Certain minor exceptions were noted but this had no overall impact to the study
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Species:
other: reconstituted huma epidermis model
Strain:
not specified
Details on test animals and environmental conditions:
not applicable
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
not applicable
Duration of treatment / exposure:
60 minutes
Observation period:
The skin irritation potential of the test substance was evaluated by measuring the relative cell viability in treated tissues at 42-hour post-exposure
incubation after a 60-minute exposure to the test substance.
Number of animals:
not applicable
Details on study design:
Upon receipt of the EpiDerm™ (MatTek Corporation), the solutions were stored as indicated by the manufacturer. The EpiDerm™ tissues were stored at 2-8ºC until used. On the day of receipt, an appropriate volume of fresh EpiDerm™ EPI-100-NMM Maintenance Medium was pre-warmed to room temperature. Nine-tenths (0.9) mL of Maintenance Medium were aliquotted into the appropriate wells of each 6-well plate, which were pre-labeled to indicate the test or control substance. Each EpiDerm™ tissue was inspected for air bubbles between the agarose gel and Millicell® insert prior to opening the sealed package. There was no tissue with air bubbles accounting for greater than 50% of the Millicell® area used in the assay. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. An appropriate number of tissues were removed from the 24-well shipping plate and transferred to the 6-well plate containing Maintenance Media. The plates were placed into the incubator at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture
conditions) for 60±5 minutes. At the end of the incubation period, the inserts were transferred into wells containing fresh Maintenance Medium and incubated overnight (18 ±3 hours) to acclimate the tissues.

The test substance was added to a 1.0 mg/mL MTT (Sigma) solution in warm MTT Addition Medium to assess its ability to directly reduce MTT. Approximately 25 mg of the test substance were added to 1 mL of the MTT solution, and the mixture was incubated in the dark at 37ºC for approximately one hour. The negative control, 30 μL of sterile, calcium and magnesium free Dulbecco’s Phosphate Buffered Saline (CMF-DPBS), was tested concurrently. If the MTT solution color turned blue/purple, the test substance was presumed to have reduced the MTT. The test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, was not observed to directly reduce MTT in the absence of viable cells.

Prior to performing the assay, the compatibility of the test substance with the nylon mesh was evaluated. Nylon meshes (Elko) were placed on a slide and 30 μL of the test substance was applied. A negative control, 30 μL of sterile, Calcium and Magnesium Free Dulbecco’s Phosphate Buffered Saline (CMF-DPBS) (Gibco, Lot # 1165768), was tested concurrently. The slides holding the treated meshes were placed into a Petri dish and incubated at standard culture conditions for 60±1 minutes. Using a microscope, each mesh was checked after 60 minutes of exposure to assess any interaction between the test substance and the mesh.
The test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, was not observed to interact with the nylon mesh, and therefore a nylon mesh was used to aid in the spreading of the test substance after dosing the EpiDerm™ tissues.

The pH of the test substance was measured using pH paper (EMD Chemicals Inc.). Initially, the test substance was added to pH paper with a 0-14 pH range in 1.0 pH unit increments to approximate a narrow pH range. Next, the test substance was added to pH paper with a narrower range of 5-10 pH units with 0.5 pH unit increments, to obtain a more accurate pH value.

The definitive assay included a negative control and a positive control. The negative control was 30 μL of 1 sterile, CMF-DPBS (Gibco, Lot # 1165768) and the positive control was 30 μL of 5% Sodium Dodecyl Sulfate (SDS) (Sigma). The 5% SDS was prepared by adding SDS powder (Sigma, Lot 069k0344v) in sterile deionized water (Quality Biological, Lot 719494). Both the positive and negative controls were tested in triplicate, and at the same exposure time as the test substance (60±1 minutes).

Definitive Test - The test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, was tested in one valid definitive trial. After the overnight incubation for 18±3 hours, the 6-well plates containing the EpiDerm™ tissues were removed from the incubator and placed at room temperature for at least 5 minutes prior to dosing. The EpiDerm™ tissues were treated in triplicate with the test substance for 60±1 minutes. Thirty microliters of the test substance were applied to each of three tissues at 1 minute intervals per tissue. A nylon mesh was placed gently over the dose to spread the test substance. If necessary, the mesh was gently pressed down to ensure even spreading. The EpiDerm™ tissues were tested in triplicate with the positive or negative control for 60±1 minutes. Thirty microliters of each control were applied to each of three tissues at 1 minute intervals per tissue. Immediately after control administration onto the tissue, a nylon mesh was placed gently over the dose to spread the negative and positive controls. The plates with dosed tissues were kept in the laminar flow hood until the last tissue was dosed. After the last tissue was dosed, all of plates were transferred to the incubator for 35±1 minutes at
standard culture conditions. After 35 minutes, all of the plates were removed from the incubator, placed into the laminar flow hood and kept at room temperature until the exposure period was completed for the first dosed tissue.

After 60 ± 1 minutes exposure, the EpiDerm™ tissues were rinsed with sterile, CMF-DPBS by filling and emptying the tissue insert 15 times. A stream of CMF-DPBS was directed onto the tissue surface. For the control substances where a mesh was used, the mesh was carefully removed with forceps (if necessary) after the 5th rinse. After the removal of the mesh, the rinsing procedure of the tissue continued for 10 times. After the 15th rinse, each of the 3 inserts per treatment group (test substance, positive, and negative control) was completely submerged, gently swirled, and the rinse media were added to a beaker containing approximately 150 mL of CMF-DPBS and specifically assigned/labelled for each treatment group; this procedure was repeated three times for each insert of each treatment group. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile CMF-DPBS from the wash bottle, and the excess CMF-DPBS was decanted. The bottom of the tissue insert was blotted on sterile paper towels and the tissue surface was carefully blotted with sterile cottontipped applicators to remove any excess moisture. The tissue surface was visually observed for residual test substance or excess moisture using a dissecting scope. Once the rinsing was complete, the tissue inserts were transferred to new 6-well plates containing 0.9 mL of fresh Maintenance Medium. The tissues were then placed into the incubator at standard culture conditions for a post-treatment expression incubation of 24±1 hours.

After the 24±1 hour post-treatment expression incubation, the 6-well plates were removed from the incubator. The tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh Maintenance Medium warmed to approximately 37ºC. The tissues were placed back into the incubator for an additional 18±1 hours post-treatment incubation at standard culture conditions.

MTT Test - A 1.0 mg/mL solution of MTT prepared in warm MTT Addition Medium was prepared no more than 2 hours before use. Three hundred microliters of MTT solution were added to each designated well of a pre-labeled 24-well plate. At the end of the 42±2 hour incubation period, the EpiDerm™ tissues were blotted on sterile paper towels to remove any excessive moisture and then transferred to the appropriate well containing 0.3 mL of MTT solution. The plates were incubated at 37±1ºC for approximately 3±0.1 hours at standard culture conditions. After the MTT incubation, the EpiDerm™ tissues were submerged into 150 mL of CMF-DPBS, swirled, and the rinse media dumped out of the Millicell®. This process was performed three times. The tissues were then blotted on sterile paper towels, and transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were covered with paraffin film, and shaken for at least 2 hours at room temperature to extract the MTT. At the end of the extraction period, the inserts were pierced with forceps and gently agitated up and down in their individual wells containing the isopropanol used for extraction. The tissue was then lifted to allow the extract to flow back into the well from which the insert was removed, and the insert was discarded. The extract solutions were mixed (homogenized by pipetting up and down three times) and two x 200 μL aliquots were transferred to the appropriate wells of a 96-well plate. Six x 200 μL aliquots of isopropanol were added to the wells designated as blanks. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader, with AUTOMIX function selected.
Irritation / corrosion parameter:
other: other: % viability
Value:
11.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 minutes and a 42 hour post exposure incubation. Reversibility: no data. (migrated information)
Other effects / acceptance of results:
The skin irritation potential of the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, was evaluated by measuring the relative cell viability in treated EpiDerm™ tissues after a 60-minute exposure and a 42-hour post-exposure incubation.
The mean OD570 of the negative control, CMF-DPBS, was 2.074. The mean viability of the positive control, 5% SDS, was 5.11%. The standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates was < 18% for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid.
The test substance was not observed to directly reduce MTT in the absence of viable cells.
Based upon the results of this assay, the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, was predicted to be irritating, and thus would require an R38 label.

SIT Results Using the EpiDerm™ Skin Model

Test substance  Concentration  % viable  R38 irritant  pH 
1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin  Undiluted  11.1  yes  5.5 
 5% SDS (positive control) NA  5.11  yes  NA 
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Based upon the results of this assay, the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, was predicted to be a skin irritant according to the prediction model validated by ECVAM, and endorsed by the ECVAM Scientific Advisory Committee (ESAC) at its 29th meeting, held on 4-5th November 2008. The protocol met the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439). Accordingly, the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, would be labeled a skin irritant, “R38” within the context of the European Risk Phrase system or a skin irritant, Category 2 as per the Guidance to Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures (2012)
Executive summary:

The MatTek Corporation’s EpiDerm reconstituted human epidermis model was used to assess the potential skin irritation of the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin (1,4-CHDM DGE). The skin irritation potential of the test substance was evaluated by measuring the relative cell viability in treated tissues at 42-hour post-exposure incubation after a 60-minute exposure to the test substance, according to the Protocol for: In vitro EpiDerm™ skin irritation test (EPI-200-SIT) evaluated and validated by the European Centre for the Validation of Alternative Methods (ECVAM). The protocol met the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439).

The test substance, 1,4-Cyclohexanedimethanol, reaction products with epichorohydrin, resulted in a 11.1% cell viability and was predicted to be a skin irritant. Results of the positive control and negative control met the criteria of a valid assay. Accordingly, the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, would be labeled a skin irritant, “R38” within the context of the European Risk Phrase system or a skin irritant, Category 2 as per the Guidance to Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures (2012).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
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 437 and in accordance with the Principles of Good Laboratory Practices (GLP)
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Remarks:
Minor exceptions which did not have any adverse impact on the study were noted
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
not applicable
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
750 µl
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
The liquid test substance 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin was tested neat (i.e., undiluted, as received). An aliquot of 750 μL of the test substance, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. Three corneas were incubated in the presence of the positive control at 32 ± 1ºC for 10 minutes. Three corneas were incubated in the presence of the negative control at 32 ± 1ºC for 10 minutes. Five corneas were incubated in the presence of the test substance at 32 ± 1ºC for 10 minutes. After the 10-minute exposure time, the control (both positive and negative) or test substance treatments were removed. The epithelial side of the corneas was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test substances. As residual test substance was observed on the corneas and it appeared to be oily, one additional rinse with Complete MEM (with phenol red) was performed on all corneas to attempt to remove the residual test substance. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior chambers were refilled with fresh Complete MEM (without phenol red) and an opacity measurement was performed. The corneas were returned to the incubator for approximately 2 hours after which a final measure of opacity was obtained.

After the final opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was filled with fresh Complete MEM and 1 mL of a 4 mg/mL fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1ºC. At the end of the 90-minute incubation period, the medium was removed from the posterior chamber and placed into tubes numbered to correspond with chamber number. Aliquots of 360 μL from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader. If the OD490 value of a control or test substance sample was 1500 or above, a 1:5 dilution of the sample was prepared in Complete MEM without phenol red (to bring the OD490 value within the linear range of the plate reader). A 360 μL sample of each 1:5 dilution was transferred to a clean specified well on the 96-well plate. The plate was read again.
Irritation parameter:
in vitro irritation score
Remarks:
mean
Run / experiment:
10 min exposure, undiluted
Value:
-1.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The in vitro score obtained for the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, was -1.2. The results of the positive control fell within two standard deviations of the historical mean (within a range of 39.2 to 63.9), the assay was considered valid.
Other effects:
not applicable

BCOP Results

Test substance 

Conc. 

Exposure time 

Mean opacity value 

Mean OD490 value 

In vitro score 

pH 

1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin 

Undiluted 

10 minutes 

-1.3 

0.006 

-1.2 

5.0 

Ethanol 

10 minutes 

42.0 

0.747 

53.2 

- 

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro score obtained for the test substance was -1.2. Based upon the results of the study, the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, is not classified as an eye corrosive or a severe eye irritant (Globally Harmonized System - GHS Category 1) according to the current OECD test guideline (OECD TG 437).
Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy/toxicity of the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin (1,4-CHDM DGE). Bovine corneas, obtained as a by-product from freshly slaughtered animals, were mounted in special holders and exposed to the test substance for 10 minutes. An in vitro score was determined for the test substance based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. The in vitro score obtained for the test substance was -1.2. Based upon the results of the study, the test substance, 1,4-Cyclohexanedimethanol, reaction products with epichlorohydrin, is not classified as an eye corrosive or a severe eye irritant (Globally Harmonized System - GHS Category 1) according to the current OECD test guideline (OECD TG 437). Furthermore, the test substance would be classified as a non-irritant to eye according to the classification system established by Vanparys et al., where a substance that induces an in vitro score between 0 and 3 would be predicted to be a non-irritant to eye.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

The potential skin irritation of CHDM DGE was assessed using the EpiDerm ™ reconstituted human epidermis model (Costin and Krcha, 2013). The protocol met the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439). The test substance, 1,4-Cyclohexanedimethanol, reaction products with epichorohydrin, resulted in a 11.1% cell viability and was predicted to be a skin irritant. Results of the positive control and negative control met the criteria of a valid assay.

Accordingly, CHDM DGE would be labeled a skin irritant, “R38” within the context of the European Risk Phrase system or a skin irritant, Category 2 as per the Guidance to Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures (2012).          

Additional dermal studies reported in 1982 with test materials differing compositions that are not entirely clear. Importantly, the test material in these studies differ from that in current use, is not relevant, and the studies were disregarded.

           Eye irritation

The potential ocular irritancy/toxicity of CHDM DGE was assessed using the Bovine Corneal Opacity and Permeability Assay (BCOP) (Costin and Sly, 2013). The protocol met the requirements of the OECD guideline, “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” (TG 437). The in vitro score obtained for the test substance was -1.2.

Based upon the results of the study, CHDM DGE is not classified as an eye corrosive or a severe eye irritant (Globally Harmonized System - GHS Category 1). Furthermore, the test substance would be classified as a non-irritant to eye according to the classification system established by Vanparys et al., where a substance that induces an in vitro score between 0 and 3 would be a non-irritant to the eye.

An additional eye irritation study was reported in 1982. While the results in this study were generally consistent with the key eye irritation study, the study report for this study does not contain sufficient information about the test material composition to permit a meaningful evaluation of the study results. Importantly, the test material in this study differs from that in current use, is not relevant, and the study was disregarded.


Justification for selection of skin irritation / corrosion endpoint:
GLP and OECD 439 guideline study.

Justification for selection of eye irritation endpoint:
GLP and OECD 437 guideline study

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Skin irritation

Based upon the skin irritation results, CHDM DGE would be labeled a skin irritant, “R38” within the context of the European Risk Phrase system or a skin irritant, Category 2 as per the Guidance to Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures (2012). 

Eye irritation         

Based on the eye irritation study results, CHDM DGE is not classified as an eye corrosive or a severe eye irritant (Globally Harmonized System - GHS Category 1). Furthermore, the test substance would be classified as a non-irritant to eye according to the classification system established by Vanparys et al., where a substance that induces an in vitro score between 0 and 3 would be a non-irritant to the eye.