Cyclohex-1,4-ylenedimethanol

Specific investigations: other studies

Administrative data

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

4 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 440 and GLPs.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Organisation for Economic Cooperation and Development (OECD) Guideline 440 for Testing of Chemicals- The Uterotrophic Bioassay in Rodents: A Short-term Screening Test for Oestrogenic Properties, 16 October 2007

Deviations:

no

Principles of method if other than guideline:

This study evaluated the ability of a mixture of 1,4-cyclohexanedimethanol (CHDM), 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD), and dimethyl terephthalate (DMT) to demonstrate or mimic biological activities consistent with agonism of natural estrogens. Animals received a combined dose of TMCD/CHDM in deionized water and then immediately a separate preparation of DMT in 0.5% methylcellulose. Both vehicles were prepared for administration to the control animals. Doses were administered per os to six groups of 10 ovariectomized female rats once daily for three consecutive days (Days 0-2). Dosage levels were 0 (solvent control), 0.001, 0.01, 0.1, 1, and 10 mg/kg bw/day, administered at a dosage volume of 5 mL/kg. The solvent control group received both vehicles at a dosage volume of 5 mL/kg . A positive control group composed of 10 ovariectomized females received 0.2 mg/kg bw/day of the estrogenic positive control agent 17α-Ethynylestradiol (EE) in corn oil on a comparable regimen, at a dosage volume of 10 mL/kg. Animals were observed for clinical signs and were weighed daily. Twenty-four hours after the last dose was administered, animals were euthanized, but no necropsies were conducted. The uteri were observed macroscopically, harvested, and wet and blotted uterine weights were recorded. The luminal fluid weight was calculated by subtracting the blotted uterine weight from the wet uterine weight. The uteri were preserved in 10% neutral buffered formalin for possible further microscopic evaluation.

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

toxicity to reproduction / fertility

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohex-1,4-ylenedimethanol

EC Number:

203-268-9

EC Name:

Cyclohex-1,4-ylenedimethanol

Cas Number:

105-08-8

Molecular formula:

C8H16O2

IUPAC Name:

cyclohexane-1,4-diyldimethanol

Constituent 2

Reference substance name:

[4-(hydroxymethyl)cyclohexyl]methanol

IUPAC Name:

[4-(hydroxymethyl)cyclohexyl]methanol

Constituent 3

Reference substance name:

Cyclohexane-1,4-dimethanol; 1,4-Bis(hydroxymethyl)cyclohexane; CHDM

IUPAC Name:

Cyclohexane-1,4-dimethanol; 1,4-Bis(hydroxymethyl)cyclohexane; CHDM

Details on test material:

1,4-Cyclohexanedimethanol (CHDM)
-Test material (as cited in report): 1,4-Cyclohexanedimethanol
-CAS number: 105-08-8
-Lot number: X30468-141
-Physical description: Clear, colorless liquid
-Purity: 99.87%
-Storage: Room temperature:
-Stability: Official stability studies were not conducted but the test substance is believed to be stable under the indicated conditions.

2,2,4,4-Tetramethyl-1,3-Cyclobutanediol (TMCD)
-Test material (as cited in report): 2,2,4,4-Tetramethyl-1,3-Cyclobutanediol
-CAS number: 3010-96-6
-Lot number: TS060513
-Physical description: White powder
-Purity: 100%
-Storage: Room temperature:
-Stability: Official stability studies were not conducted but the test substance is believed to be stable under the indicated conditions.

Dimethyl Terephthalate (DMT)
-Test material (as cited in report): Dimethyl Terephthalate
-CAS number: 120-61-6
-Lot number: X30468-142
-Physical description: White powder
-Purity: 99.98%
-Storage: Room temperature:
-Stability: Official stability studies were not conducted but the test substance is believed to be stable under the indicated conditions.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

Test animals:
-Source: Charles River Laboratories, Inc., Portage, MI
-Sex: ovariectomized female
-Age at receipt: approximately 50 days
-Age at study initiation: approximately 60 days
-Acclimation period: minimum of 11 days
-Weight at study initiation: 198-274 g
-Housing: individually housed in clean, stainless steel wire-mesh cages suspended above cage-board
-Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum
-Water: reverse osmosis-purified, ad libitum
-Method of animal identification: unique Monel® metal ear tag
-Method of animal distribution: Computer program which randomized the animals based on stratification of body weights in a block design with individual body weights at randomization within ± 20% of the mean.

Environmental Conditions:
-Temperature: 21.3 - 21.4 °C
-Humidity: 35.5-42.9%
-Photoperiod: 12-hour light/12-hour dark photoperiod
-Air exchanges: 10 fresh air exchanges/hour

In-Life Study Dates:
-Study Initiation Date: August 5, 2008
-Experimental Start Date: August 15, 2008
-Experimental Completion Date: August 20, 2008

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Deionized water and methylcellulose for test substance and control; corn oil for positive control

Details on exposure:

1,4-Cyclohexanedimethanol and 2,2,4,4-tetramethyl-1,3-cyclobutanediol were formulated together in deionized water while dimethyl terephthalate was prepared separately in 0.5% methyl cellulose. On each dosing day, each animal received a combined dose of 1,4-cyclohexanedimethanol and 2,2,4,4-tetramethyl-1,3-cyclobutanediol followed (within 1-2 minutes) by a separate dose of dimethyl terephthalate. Doses were prepared so that the test group received 0.001, 0.01, 0.1, 1, or 10 mg/kg bw/day of each test substance. The vehicle control group received separate daily doses of 0.5% methylcellulose and deionized water. The positive control, 17α-ethynylestradiol, was dissolved in a minimal amount of ethanol and then added to corn oil to achieve the appropriate dosing formulation. The vehicles, positive control, and test substance formulations were administered to their respective groups by oral gavage once daily for 3 consecutive days on study Days 0-2. The dosage volume was 5 mL/kg for the vehicle and test substance-treated groups and 10 mL/kg for the positive control substance-treated group. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg bw/day dose.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TMCD/CHDM dosing formulations were solutions; therefore, homogeneity assessment was not required. Resuspension homogeneity and stability analyses of the DMT suspension and stability assessment of the TMCD/CHDM solution were conducted in a previous study at the same testing facility.

Quadruplicate samples (1.0 mL each) for homogeneity and concentration determinations were collected from the top, middle and bottom strata of the following:
-0, 0.2 and 2 mg/mL DMT dosing formulations and 0.05 mg/mL DMT stock solution on 13 August 2008; and
-0.0002, 0.002 and 0.02 mg/mL DMT dosing formulations on 15 August 2008.

Quadruplicate samples (1.0 mL each) for concentration determination were collected from:
-0, 0.0002, 0.002, 0.02, 0.2 and 2 mg/mL TMCD/CHDM dosing formulations and 0.05 mg/mL TMCD/CHDM stock solution on 13 August 2008.

Because initial analytical results were outside acceptable ranges, back-up samples collected from the 0.0002, 0.002 and 0.02 mg/mL DMT dosing formulations on 15 August 2008 were analyzed for concentration and homogeneity; initially processed samples were reanalyzed. Back-up samples collected from the 0.2 mg/mL TMCD/CHDM dosing formulation and the 0.05 mg/mL TMCD/CHDM stock formulation were also analyzed for concentration. All analyses were conducted by the Analytical Chemistry Department of WIL Research Laboratories, LLC using a validated high performance liquid chromatography method using ultraviolet absorbance detection.

Duration of treatment / exposure:

On each of three consecutive dosing days, animals received a single dose of the 1,4-cyclohexanedimethanol/2,2,4,4-tetramethyl-1,3-cyclobutanediol formulation followed within 1-2 minutes by a single dose of dimethyl terephthalate.

Frequency of treatment:

daily for three days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.001 mg/kg bw/day TMCD/CHDM/DMT
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.01 mg/kg bw/day TMCD/CHDM/DMT
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.1 mg/kg bw/day TMCD/CHDM/DMT
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1 mg/kg bw/day TMCD/CHDM/DMT
Basis:
actual ingested

Doses / concentrations 5

Remarks:

Doses / Concentrations:
10 mg/kg bw/day TMCD/CHDM/DMT
Basis:
actual ingested

No. of animals per sex per dose:

10 females/group

Control animals:

other: yes, concurrent vehicles

Examinations

Examinations:

Clinical Observations:
Animals were observed twice daily for moribundity and mortality. Detailed clinical observations were performed daily (prior to test substance administration) through the day of euthanasia. Animals were observed for signs of toxicity at the time of dosing (within 15 minutes) and one hour after dosing.

Body Weights:
Body weights were measured daily starting the day prior to dose administration to euthanasia.

Euthanasia:
Approximately 24 hours after the last dose, all surviving animals were euthanized with carbon dioxide.

Gross Necropsy:
Full necropsies were not performed. Macroscopic examination was limited to the uteri. The uterus (with luminal fluid) from each animal was harvested, trimmed, and weighed. Then the uterus was opened, blotted, and re-weighed. The uterus and vagina were fixed in 10% buffered neutral formalin for possible future histopathology.

Positive control:

-Test substance: 17α-Ethynylestradiol (Sigma Aldrich, Inc., St. Louis, MO)
-Lot number: 107K1454
-Purity: 99.0%
-Physical description: white powder

Results and discussion

Details on results:

NOAEL: 10 mg/kg bw/day

Mortality: No mortality was observed during the study.

Clinical Abnormalities: There were no treatment-related clinical abnormalities noted during the study.

Body Weights: No test substance-related changes were noted in any TMCD/CHDM/DMT dose group. Statistically significant lower (p<0.01) body weights were observed in the positive control animals.

Macroscopic examination:
There were no test substance-related internal findings in the uterus at any dosage level. One female in the 0.001 mg/kg bw day TMCD/CHDM/DMT dose group was observed to have a small left uterine horn that also corresponded to lower wet and blotted uterine weights.

Organ Weights:
At all TMCD/CHDM/DMT dosage levels, there were no statistically significant differences from the vehicle control group. In the positive control group, mean wet and blotted uterine weights and calculated mean luminal fluid weight were significantly (p<0.01) higher compared to the vehicle control group.

Any other information on results incl. tables

Dosing Formulations:

Most of the analyzed dosing formulations were within acceptable ranges for concentrations and/or homogeneity based on the protocols used. In the absence of any test substance-related effects at any of the achieved dosage levels of dimethyl terephthalate, 1,3-cyclohexanedimethanol or 2,2,4,4-tetramethyl-1,3-cyclobutanediol, the out-of-specification results for the three lowest dimethyl terephthalate concentrations and a single intermediate 1,4-cyclohexanedimethanol concentration are not believed to affect the interpretation of the study results.

Applicant's summary and conclusion

Conclusions:

Under the conditions used in this assay, 1,4-cyclohexanedimethanol (CHDM), 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD), and dimethyl terephthalate (DMT) when administered together did not demonstrate or mimic biological activities consistent with agonism of natural estrogens at dose levels up to 10 mg/kg bw/day. Based on the results observed when the three chemicals were administered together, it is reasonable to postulate that each chemical, if administered separately, would exhibit similar negative results.

Based on the results of this study, 1,4-cyclohexanedimethanol (CHDM), 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD), and dimethyl terephthalate (DMT) when administered together have a low potential to cause endocrine disruption, are not expected to cause adverse effects in developing females, and are not classified for “Developmental or Reproductive Toxicity” according to GHS.

Executive summary:

In a uterotrophic assay, 1,4-cyclohexanedimethanol (CHDM), 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD), and dimethyl terephthalate (DMT) were administered together by oral gavage to groups of 10 ovariectomized female Sprague-Dawley rats once daily at dose levels of 0, 0.001, 0.01, 0.1, 1, or 10 mg/kg bw/day for three consecutive days. A positive control group composed of 10 ovariectomized females received 0.2 mg/kg bw/day of 17α-Ethynylestradiol (EE) in corn oil on a comparable dosing regimen. No test substance-related adverse effects on clinical observations, mean body weights, uterine weights (wet and blotted) and calculated luminal fluid were noted in any dose group. Statistically significant lower (p<0.01) body weights and significantly higher (p<0.01) mean wet, blotted uterine weights and calculated mean luminal fluid weight were observed for the positive control group. Under the conditions of the study, 1,4-cyclohexanedimethanol (CHDM), 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD), and dimethyl terephthalate (DMT) did not demonstrate or mimic biological activities consistent with agonism of natural estrogens at dose levels up to 10 mg/kg bw/day. There is nothing in this study to suggest that any of the test materials, if administered separately, would cause endocrine disruption or reproductive/ developmental toxicity.