7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Endpoint summary

Description of key information

Two repeated dose oral toxicity studies are available, one of which is a 90 day toxicity study and the other is a 14 day toxicity study.  The studies were conducted according to OECD guidelines 408 and 407, respectively.  The studies used male and female rats as the test species, specifically Crl:CD® (SD)IGS BR species.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 1999 to 15 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Proprietary guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

The dose sheets for the last five animals/sex in the 500 mg/kg/day group for study days 71-77 were inadvertently misplaced. Room temperature and relative humidity were inadvertently not recorded on study day 16.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

On study day 8, the 5 mg/kg/day group males were inadvertently dosed with the 50 mg/kg/day group formulation.

Principles of method if other than guideline:

Study was completed in accordance with standard OECD test guideline procedures

GLP compliance:

yes

Remarks:

Compliance Statement included.

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Cycloaliphatic Epoxy Resin ERL-4221
- Physical state: Clear, colorless, viscous liquid
- Analytical purity: 87.56%
- Impurities (identity and concentrations): Not documented
- Composition of test material, percentage of components: Based on the characterisation report for ERL-4221, the amount of test material in each formulation was adjusted using a correction factor for purity of 92.5%
- Isomers composition: 82-89% 3,4-epoxy cyclohexylmethy-3,4-epoxy cyclohexyl carboxylate
- Purity test date: Not documented
- Lot/batch No.: 87068
- Expiration date of the lot/batch: Not documented
- Storage condition of test material: stored at room temperature protected from light

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina.
- Age at study initiation: 28 days old at receipt.
- Weight at study initiation: 197 - 201g (males)
144 - 151g (females)
- Fasting period before study:
- Housing: Animals were housed three per cage by sex for three days on arrival. Following this, all animals were housed individually in clean, wire-mesh cages suspended above cage-board.
- Diet (e.g. ad libitum): Rodent LabDiet 5002 ad libitum, except during the period of fasting prior to blood and urine collections
- Water (e.g. ad libitum): Municipal water treated by reverse osmosis ad libitum.
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 to 29.0°C
- Humidity (%): 35.3-57%
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark photoperiod

IN-LIFE DATES: From: 10 January 2000 To:11 April 2000 for 13 week necropsy and 8 May 2000 forrecover y kill at week 17. Study completed on 4 October 2001.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Each test article formulation was prepared daily to ensure homogeneous suspension. The appropriate amount of the test article was weighed into a tared, pre-calibrated, labeled storage container. While the test article was slowly stirred with a magnetic stirrer, the vehicle was added in approximately 1-ml aliquots until the volume was brought to the appropriate calibration mark for the relevant dose group. During addition of the vehicle, the storage container was occasionally swirled manually. Once a uniform preparation was achieved, the test article formulations were not stirred further. The formulations were protected from light throughout the dosing procedures and were gently swirled prior to the sampling and dosing procedures.

The test article formulations were administered orally by gastric intubation via a 16-gauge stainless steel gavage cannula as a single daily dose for 91 or 92 consecutive days. A total dose volume of 5 ml/kg was administered to each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dosing formulations of Cycloaliphatic Epoxy Resin ERL-4221 (ERL-4221) in corn oil were analyzed to determine the test article concentration by gas chromatography with a mass selective detector. The compound response was considered to be linear from 9.47 to 114 μg/mL using a 1-μL injection. The dosing formulations (1 mg/mL and 100 mg/mL) were homogeneous with respect to ERL-4221. The mean concentrations for all strata (top, middle and bottom) ranged from 94.4 to 110% of the target dose concentrations. The relative standard deviations (RSDs) between strata for the low and high dosing formulations were 2.1% and 0.56%, respectively, indicating uniform dispersal of ERL-4221 in corn oil. The low and high dose formulat ons were stored at room temperature for 3 days and then analyzed to assess stability. The mean concentrations were 98.5 and 110% of the Time Zero concentrations, and therefore, the formulations were considered to be stable.

Duration of treatment / exposure:

91 or 92 days.

Frequency of treatment:

Daily administrations.

Remarks:

Doses / Concentrations:
5, 50 and 500 mg/kg/day.
Basis:
actual ingested

No. of animals per sex per dose:

The control and 500 mg/kg/day groups each consisted of 25 males and 25 females, and the 5 and 50 mg/kg/day groups each consisted of 20 males and 20 females.

Control animals:

yes, concurrent vehicle

Details on study design:

The test article was administered once daily by oral gavage to three groups (Groups 2- 4) at dose levels of 5, 50 and 500 mg/kg/day, respectively. Doses were based on an epoxy equivalent correction factor of 92.5%. A concurrent control group (Group 1) received the vehicle, Mazola ® corn oil, on a comparable regimen. The control and 500 mg/kg/day groups each consisted of 25 males and 25 females, and the 5 and 50 mg/kg/day groups each
consisted of 20 males and 20 females. A dose volume of 5 ml/kg was used for all groups.

Groups of 15 rats/sex/dose level were assigned to the primary necropsy, and 5 rats/sex/dose level (Groups 2 and 3) or 10 rats/sex/dose level (Groups 1 and 4) were assigned to a 28-day recovery period.

Parameters evaluated included clinical observations, body weights, food consumption, clinical pathology (haematology, serum chemistry and urinalysis), ophthalmology, vaginal cytology and spermatogenic endpoints. Complete necropsies were performed on all animals and selected organs were weighed. Selected tissues were examined microscopically at the primary and recovery necropsies.

Positive control:

10 animals/sex not assigned to the study from the same shipment of animals (with body weights closest to the group mean for animals placed on the study) were maintained in the animal room throughout the study. These animals were used as positive controls.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed on all animals just prior to dosing and approximately one to two hours following dosing. Each animal received a detailed physical examination weekly, beginning 10 days prior to the test article administration and just prior to the scheduled necropsy. When appropriate, explicitly defined scoring systems were used. Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded. Signs such as skin lesions and hair loss were also recorded at this time. The absence or presence of findings was recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded approximately weekly, beginning 10 days prior to test article administration. Mean body weights were calculated for each study week and mean body weight changes were calculated for the corresponding intervals. A final body weight (fasted) was recorded for each animal on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Individual food consumption was measured approximately weekly, beginning 10 days prior to test article administration (study week -2). Food consumption was not measured from the day of randomization to study day 0. Food intake was calculated as g/animal/day for the corresponding body weight intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ocular examinations were conducted on all animals prior to the initiation of dosing and during study week 12. All ocular examinations were conducted using an indirect ophthalmoscope, preceded by mydriasis.
- Dose groups that were examined: All dose groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All time points. During Weeks 5, 13 and 17
- Anaesthetic used for blood collection: No required - blod obtained from tail vein
- Animals fasted: Yes - overnight
- How many animals: 10 animals/sex/group assigned to the primary necropsy during study week 5 and on all animals assigned to the scheduled necropsies (study weeks 13 and 17).
- Parameters checked: Total Leukocyte Count, Erythrocyte Count, Haemoglobin, Haematocrit, Mean Corpuscular Volume, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Platelet count, Prothrombin time, Activate Partial Thromboplastin Time, Differential Leukocyte Count Percent and Absolute, Platelet estimate and Red Blood Cell Morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: All time points. During Weeks 5, 13 and 17
- Animals fasted: Yes - overnight
- How many animals: 10 animals/sex/group assigned to the primary necropsy during study week 5 and on all animals assigned to the scheduled necropsies (study weeks 13 and 17)
- Parameters checked: Albumin, Total Protein, Globulin, Albumin/Globulin Ratio, Total Bilirubin, Urea Nitrogen, Creatinine, Alkaline Phosphatase, Alanine Aminotransferase, Aspartate Aminotransferase, Gamma Glutamyltransferase, Glucose, Total Cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Creatine Kinase, Lactate Dehydrogenase, Direct Bilirubin, Indirect Bilirubin, Sorbitol Dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to the day blood samples were collected during Weeks 5, 13 and 17.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: Specific Gravity, PH, Urobilinogen, Total Volume, Urine Creatinine, Urine N-acetyl-β-glucosamidase, Osmolality, Color, Appearance, Protein, Glucose, Ketones, Bilirubin, Occult Blood, Leukocytes, Nitrites, Microscopy of Sediment.

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes-A complete necropsy was conducted on all animals. The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera.
HISTOPATHOLOGY: Yes

A full standard guideline tissue list was preserved at necropsy for histopathological examination. All tissues were examined for the control and high dose group killed at week 13. Microscopic examination was also conducted on the kidneys, liver, lungs, nasal tissues and gross lesions in the 5 and
50 mg/kg/day groups at the primary necropsy.

For animals in the recovery groups, microscopic examination was conducted on the liver in the control, 50 and 500 mg/kg/day groups and nasal tissues in the control, 5, 50 and 500 mg/kg/day groups.

The slides for nasal turbinate sections were peer-reviewed for determination of the nature and significance of the the epithelial degeneration (and regeneration following the recovery phase)

Other examinations:

Determination of Oestrous Cycle: Vaginal smears for determination of the stage of oestrus were obtained from all females once daily beginning 22 or23 days prior to the primary necropsy. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P]) beginning 22 or 23 days prior to the primary necropsy. The final vaginal smear for each female was collected on the day of necropsy.

Spermatogenic Analysis - Motility/Viability Assessment, Morphology Assessment and Enumeration of Epididymal and Testicular Sperm numbers and Sperm Production Rate - investigations completed immediately following euthanasia. A diiferential count of 200 spermatozoa/rat was used to determine morphological differences. The left testis and epididymis from each male at the primary necropsy evaluated for sperm numbers and sperm production rate using the method described by Blazak et al

Organ Weights
The following organs were weighed from all animals at the scheduled
necropsies:
Adrenals
Brain
Epididymides (total and cauda)
Heart
Kidneys
Liver
Ovaries (with oviducts)
Prostate
Spleen
Testes
Thymus
Thyroid glands with parathyroids
Uterus (with cervix).
Paired organs were weighed together. Organ to final body weight and organ to brain weight ratios were
calculated.

Statistics:

All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5% comparing the control group to the treatment groups (by sex). Body weight, body weight change, food consumption, clinical data, oestrous cycle data, organ weight data, epididymal and testicular sperm numbers and sperm production rates were subjected to a oneway analysis of variance (ANOVA), followed by Dunnett's test if the ANOVA revealed statistical significance. The percentage of motile spermatozoa and the percentage of sperm with normal morphology were analyzed by the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences, followed by the Mann-Whitney U-Test comparing the control and test article-treated groups if the ANOVA revealed statistical significance (p<0.05).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test article-related clinical observations were noted during the recovery period.

Mortality:

mortality observed, treatment-related

Description (incidence):

No test article-related clinical observations were noted during the recovery period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See results below

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See results below

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

See results below

Haematological findings:

no effects observed

Description (incidence and severity):

See results below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See results below

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See results below

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See results below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See results below

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See results below

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Test article-related clinical observations consisted of evidence of increased salivation (clear material around the mouth and on the forelimbs and ventral neck) and yellow material on various body surfaces (urogenital area, anogenital area, hindlimbs and ventral neck/trunk) in the 500 mg/kg/day group males and females. These findings were primarily noted at the 1-hour post-dosing observation, and less frequently noted prior to dosing and during detailed physical examination. Evidence of increased salivation and yellow material on various body surfaces were first noted on study days 8 and 9, respectively, and, once noted, were observed sporadically throughout the remainder of the dosing period. No test article-related clinical observations were noted during the recovery period.

All animals survived to the scheduled necropsies (study weeks 13 and 17).

BODY WEIGHT AND WEIGHT GAIN
Mean body weights were lower in the 500 mg/kg/day group males throughout the study when compared to the control group. The lower mean body weights were considered to be related to test article treatment. At the end of the dosing period (study week 13), mean body weight in the 500 mg/kg/day group males was approximately 7% lower than the control group. For males, weight gain was significantly reduced in the 500 mg/kg/day group during study week 5 to 6 and study week 11 to 12. There were no clear treatment-related differences in mean absolute body weight for the females in any group.
Some statistically significant differences were noted when body weight gains for the control and test article-treated groups were compared; however,
these differences were not considered test article-related. Weight gain for males in the 50 and 500 mg/kg/day groups was lower than the control group for the week preceding the initiation of dosing. However, as these differences did not result in significantly lower mean body weights compared to the controls at the time of the first dose, they were considered to be random biological variability.
For females, a decrease in mean weight gain (approximately 19% less than the control group) was noted for the high dose group during the first week of dosing. This difference from the control group was considered to be statistically significant and resulted in a reduction in mean cumulative weight gain (statistically significant at study weeks 1 and 3) which remained throughout the dosing period.
Overall weight gain during the recovery period was similar across doses for males and females and there was no clear evidence of the high dose group males returning to control mean level.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No test article-related changes were noted in food consumption. However, some statistically significant differences were noted when the control and treated groups were compared. A slight decrease in food consumption was observed in the 500 mg/kg/day group females during study week 0 to 1 when compared to the control group. In addition, higher food consumption was observed in the 500 mg/kg/day group males during study weeks 3 to 4 and 5 to 6 and in the 50 mg/kg/day group males during study week 12 to 13. These differences from the control group were slight and no trends were apparent. Therefore, these changes were not attributed to test article administration. There were no other remarkable changes in food consumption during the dosing or recovery

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not examined

OPHTHALMOSCOPIC EXAMINATION
No oculopathic changes indicative of a test article-related effect were observed at any dose level.

HAEMATOLOGY
There were no test article-related changes in haematology parameters in any treatment group. However, several statistically significant differences were observed when the control and test article-treated groups were compared. At the study week 5 evaluation, mean white blood cell count was increased in the 500 mg/kg/day group males, mean red blood cell count was increased in the 50 mg/kg/day group males and mean absolute lymphocyte counts were increased in the 5 mg/kg/day group males.

At the study week 13 evaluation, increased mean white blood cell count was observed in the 5 mg/kg/day group males, decreased mean MCHC was noted in the 500 mg/kg/day group males and mean prothrombin times were increased in the 50 and 500 mg/kg/day group males and the 50 mg/kg/day group females. Mean absolute lymphocyte count was higher in the 5 mg/kg/day group males and mean percent and absolute neutrophil counts were lower and mean percent lymphocyte counts were higher in the 50 and 500 mg/kg/day group females.

All of the changes observed occurred sporadically, were without apparent dose-related response, were of minor magnitude and were inconsistent between the sexes. Consequently, the haematologic differences observed were considered to be of normal biological variation and were not test article related.

CLINICAL CHEMISTRY
Changes in serum chemistry parameters considered to be related to the toxicity of the test article consisted of elevated urea nitrogen and phosphorus levels and reduced cholesterol levels (statistically significant in the 500 mg/kg/day males only) in the 50 and 500 mg/kg/day groups and elevated direct bilirubin and sorbitol dehydrogenase levels in the 500 mg/kg/day group.
Mean urea nitrogen (BUN) levels were increased in the 50 and 500 mg/kg/day group males and females at the study week 5 and 13 evaluations when compared to the control group.
Mean phosphorus levels were increased in the 500 mg/kg/day group males and females at the study week 5 evaluation (statistically significant for males only) and in the 50 and 500 mg/kg/day group males and females at the study week 13 evaluation when compared to the control group (statistically significant).
Mean creatine kinase levels were decreased in the 500mg/kg/day group in males at the study week 5 evaluation and in a dose related manner at the study week 13 evaluation when compared to the control group. These decreases were determined to be statistically significant.
Mean serum cholesterol concentrations were decreased in the 50 and 500 mg/kg/day males at the study week 5 and 13 evaluations when compared to the control group, however, difference was only statistically significant in the 500mg/kg/day group only. In females, mean serum cholesterol concentrations were decreased in the 50 mg/kg/day group at the 13 week evaluation.
Mean direct bilirubin and sorbitol dehydrogenase levels in the 500 mg/kg/day group males and females were increased (usually statistically significant) at the study week 5 and 13 evaluations when compared to the control group.
In addition to the above observations, several statistically significant differences were observed when the test article-treated groups were compared to the control group. In males, mean calcium, sodium and lactate dehydrogenase levels differed from the control group values at different study intervals in various groups. Mean creatine kinase levels in the 5 and 50 mg/kg/day group males were decreased at the study week 13 evaluation when compared to the control group.
In females, mean total protein, globulin, bilirubin (indirect and total), glucose, calcium and potassium levels differed from the control group values at different study intervals in various groups. However, these changes observed in females occurred sporadically, did not occur in a dose related manner, were of minor magnitude, did not display consistency between sexes and were biologically irrelevant. As no macroscopic or microscopic changes
changes correlated with these serum chemistry alterations, the serum chemistry differences were considered to be spurious, the result of biological variation and not test article-related.

There were no treatment-related effects on serum chemistry parameters following the recovery period. The only statistically significant differences from the control group at study evaluation week 17 (recovery period) consisted of increased mean potassium and indirect bilirubin levels for the 5mg/kg/day group females and an increased mean lactate dehydrogenase value for the 500mg/kg/day males. These changes however, did not occur in a dose-related manner, were inconsistent between sexes, were biologically irrelevant and/or did not have any corresponding macroscopic or microscopic findings.

URINALYSIS
Changes in urinalysis parameters considered to be related to test article treatment consisted of decreased mean urine pH in the 500 mg/kg/day group males and females and decreased mean urine creatinine levels in the 500 mg/kg/day group males. Urine pH was decreased in the 500 mg/kg/day group males and females at the study week 5 and 13 evaluations (usually statistically significant). Urine creatinine levels for males in the 500 mg/kg/day group were decreased at the study week 5 and 13 evaluations (statistically significant). However, when adjusted for time and body weight, the effect was not apparent.
When the control and test article treated groups were compared, some statistically significant differences were observed. A lower mean creatinine was observed in the 5mg/kg/day group males at study week 5 evaluation, higher mean urobilinogen was noted in the 500 mg/kg/day group males at the study week 5 evaluation and higher mean osmolality was noted in the 500 mg/kg/day group females at the study week 13 evaluation. However, these changes were not considered to be test article related as they were only noted at a single evaluation, were not consistent between the sexes and there were no correlating microscopic findings.
During the recovery period, all urinalysis values for all dose groups of both sexes were similar to the control group values.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
Test article-related organ weight changes consisted of higher mean liver weights (usually statistically significant) in the 50 and 500 mg/kg/day group males and females and higher mean kidney weights in the 500 mg/kg/day group males and females at the study week 13 necropsy.
In the 50 and 500mg/kg/day males, and females, mean absolute and relative liver weights were increased at necropsy in week 13. The differences from the control group were statistically significant with the exception of absolute liver weight and liver weight relative to brain weight.
In males and females in the 500mg/kg/day dose group, mean absolute and relative kidney weights were increased at study week 13 necropsy in comparison to the control group. These differences observed were considered to be statistically significant compared to the control group.
When the control and test-article treated groups were compared, several statistically significant differences were observed. Mean absolute prostate weight was decreased in the 5mg/kg/day group males and mean thryoid weight relative to final body weight was increased in the 500mg/kg/day group males at the study week 13 necropsy. As no dose-related trends were apparent and there were no correlating macroscopic or microscopic changes, these organ weight changes were considered to be a result of normal biological variation and unrelated to test article administration.

No test article-related differences in organ weights were observed for any dose group of both sexes following the recovery period. Thymus weights were significantly increased in the 50mg/kg/day males, however, this increase was not considered to be treatment related as a similar effect was not evaluated at any previous time-point evaluation or at any higher concentrations.

GROSS PATHOLOGY
Test article-related macroscopic findings were noted in the liver of 3/15 males in the 500 mg/kg/day group at the study week 13 necropsy.
Pale livers were observed in 3/15 males in the 500 mg/kg/day group. Pale livers were not noted in any other group of males, including the control
group, and were not noted in any female group. No other test article-related gross findings were observed at either necropsy. All other macroscopic changes were considered to be spontaneous and/or incidental in nature and unrelated to test article administration.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test article-related microscopic changes were observed in the liver of the 50 and 500 mg/kg/day group males and females at the primary necropsy and in the nasal tissues of 50 and 500 mg/kg/day males and females at the study week 13 (primary) and 17 (recovery) necropsies.
At the study week 13 necropsy, in both males and females in the 50 and 500mg/kg/day dose groups, fine hepatocellular vacuolation, in a periportal distribution, was observed with increased incidence.
The primary lesion observed in the nasal tissues in the 50 and 500 mg/kg/day group males and females were degeneration of olfactory epithelium. Some of these olfactory epithelial degenerative changes persisted at the recovery evaluation in males and females at the aforementioned dose groups, however, some regeneration of the olfactory tissue was also observed.

HISTORICAL CONTROL DATA (if applicable)
No data

OTHER FINDINGS
Oestrous Cycle Data: No test article-related changes in estrous cycle were observed.
Spermatogenic Evaluations: No test article-related effects on spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate and sperm motility and morphology) were observed at any dose level.

Dose descriptor:

NOEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

The NOAEL of 5 mg/kg bw/day was determined based on systemic effects observed in the high dose group – increased liver and kidney weights, reduced bodyweight gains and lower food consumption, which were consistent with similar findings observed at the same dose level in the pre-natal developmental toxicity study. The liver effects are considered to be adaptive, indicating a response to a mild toxic insult; the changes were reversible within the recovery phase and showed no dose response relationship. The renal weight change was not associated with any clinical pathology or microscopic changes that could clarify the cause of this effect. Kidney weight changes were also restricted to the high dose group.

The observation of nasal epithelial degeneration extended to the intermediate dose group (2/15 males and 3/15 females affected by minimal degeneration). The effects were more pronounced and in greater incidence in the high dose group. The unusual nature of the response – nasal damage caused after oral cannula administration – appears to have been raised by the study director and the nasal tissue slides were specifically subjected to peer review by a second pathologist. While the pathologists agreed a form of wording to describe the pathology, it is clear from the pathology statement that identification of the cause or the toxicological significance of this nasal finding was not agreed by the pathologists. However, no additional investigations have been disclosed to provide any new information that could address the mode of activity/cause of induction of nasal change. 

It has been postulated that refluxing of the oral dose could affect the nasal turbinates but there is no corroborating evidence in the report to suggest any difficulties during the dose administration or recording of any clinical signs post-dosing that could suggest the animals were discomforted or were involuntarily removing the dose depot by reflux action. Nasal epithelial degeneration is normally associated with inhalation of toxic fumes. The vapour pressure of the substance is 2 x 10E-3Pa, and the administered dose was formulated in corn oil. It is considered highly improbable that the high dose animals were consistently exposed to toxic/caustic vapours from this oral preparation (the acute inhalation study showed no indication of nasal damage, nasal irritation or production of nasal exudates when animals were exposed to a 5.5 mg/L atmosphere for 4 hours nose-only exposure). Since it is not possible from the available study data to determine a mode of action or aetiology for the nasal change, conclusions regarding the systemic or local nature of the effect cannot be drawn.

The nasal epithelial changes were effectively the critical data on which the NOAEL derivation was based. The cause was not established, the persistence and limited recovery can be gauged by the incidence of degenerative and regenerative change present at termination following the recovery phase. It has been assumed that the nasal change is a systemic effect and extended in a dose related manner to affect the high dose group moderately and the intermediate group minimally and was not present in any of the low dose group animals. 

Assuming the systemic derivation of the change at 50 and 500 mg/kg bw/day means the oral NOAEL of 5 mg/kg bw/day is the point of departure for derivation of systemic DNEL values. In view of the likelihood of the nasal findings being idiopathic or localised in derivation, use of the low dose NOAEL is highly conservative and ensures any tier 1 risk assessments will err on the precautionary side.

Conclusions:

Based on the results of this study, the no-observed-effect level (NOEL) for oral administration of Cycloaliphatic Epoxy Resin ERL-4221 to rats for a
minimum of 90 days was 5 mg/kg/day for both males and females. Based on these results, the test substance does not require classification according to Directive 67/548/EEC or Regulation 67/548/EEC.

Executive summary:

In the study conducted by Padgett (2001), the toxicity potential of the test substance, Cycloaliphatic Epoxy Resin ERL-4221, was evaluated following 90 day oral (gavage) administration to Crl:CD (SD)IGS BR rats. The test article was adminstered once daily by oral gavage to three groups of rats at dose levels of 5, 50 and 500mg/kg/day. A concurrent control group received the vehicle, corn oil, on a comparable regimen. The control and 500 mg/kg/day groups each consisted of 25 males and 25 females, and the 5 and 50 mg/kg/day groups each consisted of 20 males and 20 females. A dose volume of 5ml/kg was used for all groups.

Observations included clinical observations, body weights, food consumption, clinical pathology (hematology, serum chemistry and urinalysis), ophthalmology, vaginal cytology and spermatogenic endpoints. Complete necropsies were performed on all animals and selected organs were weighed. Selected tissues were examined microscopically at the primary and recovery necropsies.

There were no mortalities reported. There were no test article-related changes in food consumption or hematology parameters, oculopathic changes or in the oestrous cycle or spermatogenic endpoints observed. Increased salivation and yellow material on the fur were observed primarily after dosing in the 500 mg/kg/day group males and females. Body weights of the males from the 500 mg/kg/day group were decreased, although not statistically significant, throughout the study. Following the 13 week dosing period, effects were noted in the kidney, liver and olfactory epithelium of the nasal tissues. Slight effects in absolute and relative kidney weights in the 500 mg/kg/day group and correlating serum chemistry and urinalysis changes in the 50 and 500 mg/kg/day groups were observed. There were no histopathologic changes noted in the kidneys. In the 50 and 500mg/kg/day dose groups, slight effects were observed in absolute and relative liver weights as well as liver function, accompanied by minimal to mild histopathological changes. Degeneration of the olfactory epithelium in the nasal tissues was seen in the 50 and 500 mg/kg/day group males and females but not in any of the control group animals.

Following a 28 day recovery period, the mean body weight of males in the 500mg/kg/day dose group was lower than the controls although weight gain during the same period was similar. Olfactory epithelial degeneration was observed in both males and females from the 50 and 500 mg/kg/day groups, although at a lower incidence. In addition, regenerative changes were also evident in the olfactory epithelium.

Based on the results of this study, the NOEL for oral administration of Cycloaliphatic Epoxy Resin ERL-4221 to rats for a minimum of 90 days was determined to be 5 mg/kg/day for both males and females.

Based on these results, the test substance does not require classification according to Directive 67/548/EEC or Regulation 67/548/EEC.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

reliable , guideline compliant investigation with additional recovery phase and inclusion of extra parameters to enable initial assessment of reproductive toxicity potential.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated Dose Oral Toxicity:

In the study conducted by Padgett (2001), the toxicity potential of the test substance, Cycloaliphatic Epoxy Resin ERL-4221, was evaluated following 90 day oral (gavage) administration to Crl:CD (SD)IGS BR rats. The test article was administered once daily by oral gavage to three groups of rats at dose levels of 5, 50 and 500 mg/kg/day. A concurrent control group received the vehicle, corn oil, on a comparable regimen. The control and 500 mg/kg/day groups each consisted of 25 males and 25 females, and the 5 and 50 mg/kg/day groups each consisted of 20 males and 20 females. A dose volume of 5 ml/kg was used for all groups.

Observations included clinical observations, body weights, food consumption, clinical pathology, ophthalmology, vaginal cytology and spermatogenic endpoints. Complete necropsies were performed on all animals and selected organs were weighed. Selected tissues were examined microscopically at the primary and recovery necropsies.

There were no mortalities reported. There were no test article-related changes in food consumption or haematology parameters, oculopathic changes or in the oestrous cycle or spermatogenic endpoints observed. Increased salivation and yellow material on the fur were observed primarily after dosing in the 500 mg/kg/day group males and females. Body weights of the males from the 500 mg/kg/day group were decreased throughout the study. Following the 13 week dosing period, effects were noted in the kidney, liver and olfactory epithelium of the nasal tissues. Slight effects in absolute and relative kidney weights in the 500 mg/kg/day group and correlating serum chemistry and urinalysis changes in the 50 and 500 mg/kg/day groups were observed. There were no histopathologic changes noted in the kidneys. In the 50 and 500 mg/kg/day dose groups, slight effects were observed in absolute and relative liver weights as well as liver function, accompanied by minimal to mild histopathological changes. Degeneration of the olfactory epithelium in the nasal tissues was seen in the 50 and 500 mg/kg/day group males and females but not in any of the control group animals.

The critical endpoint in this repeated dose oral toxicity study was nasal epithelial degeneration, an apparent systemic adverse effect observed in the high dose group predominantly but extending also to the intermediate dose group at much lower incidence and severity. The cause of the epithelial damage was not ascertained despite peer review of the terminal and post-recovery findings. The induction of epithelial basal cell degeneration is normally attributable to inhalation of toxic fumes but for 7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate the vapour pressure is low and vapours, particularly from the material formulated in corn oil, are considered highly unlikely to be the source of the nasal effects.

Similar nasal effects have been observed following administration of epoxides where hepatic metabolism of epoxy groups can result in dihydrodiol formation and hydrolysis of the ester groups. Systemic nasal toxicity has been observed where the generation of reactive epoxide metabolites occurs in nasal epithelium following hepatic metabolism and mediation by cytochrome P450 isozymes. The expression of nasal toxicity is influenced by relative levels of P450, epoxide hydrolase and glutathione. The effects observed in the subchronic oral toxicity study (including hepatic enzyme induction, liver enlargement and persistent nasal epithelial degeneration) are therefore considered to result from administration-route-independent, systemic exposure rather than inhalation of the test substance or exposure to reactive metabolites present or excreted in exhaled air or via volatiles released from the test substance or secondary exposure to highly volatile metabolites. While the precise mechanisms were not evaluated in the current study, the nature of the nasal effects and similarity of response when compared to other epoxy materials is strongly indicative of a systemic exposure hazard under metabolic influence.

Following a 28 day recovery period, the mean body weight of males in the 500mg/kg/day dose group was lower than the controls. Olfactory epithelial degeneration was observed in both males and females from the 50 and 500 mg/kg/day groups, although at a lower incidence. In addition, regenerative changes were also evident in the olfactory epithelium.

Based on the results of this study, the NOEL for oral administration of Cycloaliphatic Epoxy Resin ERL-4221 to rats for a minimum of 90 days was determined to be 5 mg/kg/day for both males and females.

Review of 90-day toxicity study report – Padgett 2001

ERL-4221 formulated in corn oil was administered orally (by cannula rather than gavage) to three groups – 5, 50 and 500 mg/kg bw/day.  25 rats in the control and high dose group; 20 rats in the low and intermediate dose groups. 15 rats per group were assigned to necropsy at the end of treatment (day 91 / 92) and the remaining animals were retained for a 28 day recovery/treatment free period. The microscopic examinations were completed for all groups with respect to nasal tissues and the nasal slides were reviewed by a second pathologist.

 

A small effect on male bodyweight gain was observed in the 500 mg/kg bw group (overall gain reduced by 7% compared with controls), no overall effect was noticed in females (although 19% reduction in mean weight gain occurred over week 1), or for lower dosed males. Reduced food consumption was recorded in week 1 for females dosed at 500 mg/kg bw/day. It is unclear whether these were treatment related effects or related to palatability/ inanition resulting from administration of corn oil (or possibly related to eating discomfort following cannula dosing).

No treatment related effects were noted for haematology or serum chemistry parameters – various changes were attributed to normal background variation, no consistent dose related responses and none that could be correlated with pathology findings were apparent.

No urinalysis effects were apparent, no pathological corroboration of renal weight changes in this study and no observed urinalysis or pathology change that could be linked to minor renal changes observed in the developmental toxicity study. Although various minor changes were evident in several parameters, none were consistent over several sampling occasions and were not attributed to treatment. No effects were observed on oestrous cycling. No treatment related spermatogenic effects were observed.

Macroscopic pathology – 3/15 high dose males had pale livers. No hepatic effects seen in other groups. No other treatment related macroscopic changes. 

Organ weight – higher liver weights were recorded at 50 and 500 mg/kg bw/d.  Higher kidney weights were recorded for males and females at 500 mg/kg bw/d.

Microscopic pathology – liver effects for males and females at 50 and 500 mg/kg – no similar effects in the 5 mg/kg bw group.

Changes in nasal tissues at 50 and 500 for males and females at week 13 and post recovery week 17.

Hepatocellular vacuolation incidence was increased in 50 and 500 mg/kg bw/day groups. The effects were minimal in the control, 5 and 50 mg/kg bw/d groups and mild in the high dose group and all effects in the liver had fully reversed following the recovery period.

Males and females in the 50 and 500 mg/kg bw/d groups had degeneration of olfactory epithelium. The degeneration of the nasal epithelium was characterised by the loss of sustentacular cells, vacuolation and desquamation of the neuroepithelial cells resulting in the decreased height of the olfactory epithelium. Basal cells and the underlying structures (Bowmans glands, ducts, olfactory nerve bundles and connective tissue) were not affected. Although degeneration of the olfactory epithelium persisted after the recovery period there was also evidence of regeneration.

 Week 13: liver  minimal hepatocellular vacuolation – 15/15 males and 12/15 females at 50 mg/kg bw/d
                         mild hepatocellular vacuolation – 15/15 males and 15/15 females at 500 mg/kg bw/d

            Nasal epithelium degeneration – minimal – 3/15 females; mild – 2/15 males at 50 mg/kg bw/d
                         mild – 2/15 females; moderate – 7/15 males and 11/15 females; severe 5/15 males at 500 mg/kg bw/d

 Week 17: Liver vacuolation had resolved – no incidence in any group.   Liver vacuolation is normally associated with mild liver insult and the appearance of glycogen filled vacuoles may be a beneficial adaptive change protecting against further liver damage by more severe toxic insult. The recovery of livers after 28 days treatment free and the absence of any supporting inlife, clinical pathology or macroscopic changes would indicate the liver effects were not toxicologically significant. 

 

The reported nasal epithelial degeneration appears to be a local effect - and would typically arise from inhalation of toxic fumes but in this case the low vapour pressure of the test material would suggest no inhalation exposure occurred and consequently a systemic cause may be responsible for the epithelial effects, although the cause was not determined in this investigation. Additional microscopic pathology indicates respiratory epithelial inflammation and inflammatory exudates for males dosed at 50 mg/kg bw/d (animals nos. 37338; 37343; 37375) although this was not evident in rats with epithelial degeneration and the males dosed at 500 mg/kg bw/d only displayed epithelial degeneration, no inflammatory response. In the females dosed at 50 or 500 only one had epithelial degeneration and an inflammatory response (37443).

The low incidence and minimal severity of epithelial change at 50 mg/kg, the absence of corroborating evidence for inflammatory or degenerative responses following exposure to other mucus membranes and the absence of other systemic toxic effects indicate the nasal epithelial changes to be other unclear toxicological significance – a local effect that may be related to test substance exposure, but without any clear mode of action – it is unlikely the high dose animals inhaled significantly high doses due to the low a.s. vapour pressure and the other postulated mechanism – reflux of the oral dose has no support from reported clinical observations or records of mis-dosing events/ signs of choking etc.

Acute 4h inhalation exposure to 5.5 mg/L was well tolerated with no toxicity observed and no macroscopic abnormalities detected (but microscopic examination of nasal turbinates was not conducted in this study). The vapour pressure for ERL-4221 is very low – 2x 10-3 Pa which would suggest the nasal epithelial changes did not result from inhalation of toxic vapours from the test material

The toxicological significance of the nasal effects is unclear given the route of exposure of rats was by gavage and there is no evidence for inhalation exposure or accidental exposure via refluxation or mis-dosing. As a worst case assumption, the cause of nasal effects is considered to be systemic and the NOAEL is used in the risk assessment as a highly conservative starting point from which to derive a DNEL for long term systemic inhalation exposure.

 

The NOAEL was concluded to be 5 mg/kg bw/day but on review it would appear that 5 mg//kg bw/d is a NOEL.

The liver effects observed in the 50 mg/kg bw/day group are minimal and fully reversible following cessation of treatment and do not appear to be toxicologically significant. The increased liver weight at 50 and 500 may be an adaptive response to mild toxic insult.

 

Changes were evident at the high dose, 500 mg/kg bw/day, including effects on bodyweight, food consumption liver and kidney weights in addition to the local effect on the majority of high dose group nasal turbinate epithelial degeneration. Only minimal nasal effects were observed in the group dosed at 50 mg/kg bw/day, which may be concluded to be the LOAEL in this study.

 

In the study conducted by Padgett (2000), the possible toxic effects of Cycloaliphatic Epoxy resin ERL-4221were evaluated using a 14 day dose-range finding study in Crl:CD^® (SD)IGS BR rats. The test article was administered once daily by oral gavage to four groups (Groups 2-5) each consisting of 10 males and 10 females. Dose levels were 100, 500, 750 and 1000 mg/kg/day for Groups 2-5, respectively. A concurrent control group consisting of 10 males and 10 females received the vehicle, Mazola^® corn oil, on a comparable regimen. A dose volume of 5 ml/kg was used for all groups. Parameters evaluated included clinical observations, body weights and food consumption. Complete necropsies were performed on all animals and selected organs were weighed. Selected tissues were examined microscopically.

All animals survived to the scheduled necropsy. There were no macroscopic findings attributed to the test article. Test article-related clinical observations consisted of evidence of increased salivation in the 500, 750 and 1000 mg/kg/day group males and females and yellow material on the urogenital area in the 750 and 1000 mg/kg/day group females. Dose-related decreases in body weight and weight gain were observed for males dosed at 500 mg/kg/day and above and for females in the high dose group. Test article-related decreased mean food consumption was observed in the 750 and 1000 mg/kg/day group males during the first week of the study. In males, increased mean absolute and relative liver weights were observed in the 100, 500, 750 and 1000 mg/kg/day groups. Test article-related increases in mean absolute and relative liver weights were noted for the 500, 750 and 1000 mg/kg/day group females. There was no clear dose response for these increases. These changes in liver weights correlated with an increased incidence and/or severity of periportal hepatocellular vacuolation observed microscopically in the 100 mg/kg/day group males and in the 500, 750 and 1000 mg/kg/day group males and females, although the changes in the livers of females was minimal.

Based on clinical findings (increased salivation and yellow material on the urogenital area) and decreases in body weight gains in the 500, 750 and 1000 mg/kg/day groups, decreases in food consumption in the 750 and 1000 mg/kg/day groups, and histopathological effects on liver and corresponding increases in liver weights observed in the 100, 500, 750 and/or 1000 mg/kg/day groups, the no observed- adverse-effect level for Cycloaliphatic Epoxy Resin ERL-4221 could not be determined.

The key study was considered to be the 2001 main study conducted by Padgett as it was a definitive study, the 14 -day study provided dose range-finding information to support the main study.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Sole subchronic exposure study available to address repeated exposure via oral route

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; respiratory: nose; urogenital: kidneys

Justification for classification or non-classification

Based on the results of the key study, (Padgett, 2001), the test substance, Cycloaliphatic Epoxy Resin ERL-4221, was determined to have an NOEL of 5mg/kg/day for both males and females. Based on these results, the test substance does not require classification according to Directive 67/548/EEC or Regulation 67/548/EEC - even at the next higher dose level of 50 mg/kg bw/day the effects observed in liver and kidneys were transient and reversible and did not indicate serious morphological disturbance or irreversible target organ toxicity. While dose-related effects on nasal epithelium were identified, the mode of toxic action or causation of nasal changes following oral administration via cannula were not established but the low vapour pressure for the active substance suggests the inhalation route was not the primary exposure route for eliciting the epithelial change.