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Toxicological information

Carcinogenicity

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Administrative data

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The 2-year studies in rats were begun in April 1979 and completed in April 1981. The supplemental rat studies were started in April 1980 and completed in April 1982.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Toxicology and carcinogenesis studies of technical grade diglycidyl resorcinol ether (81%pure) were conducted by administering the chemical in corn oil by gavage to groups of 50 male and 50 female F344/Nrats at doses of 25 or 50 mg/kg. A supplemental study of similar design in male and female rats (0 or 12 mg/kg)was started approximately 12 months later because of high mortality in the 50 mg/kg dose groups. Doses were administered five times per week for 103 weeks. Groups of 50 rats of each sex received corn oil by gavage on the same dosing schedule and served as vehicle controls.
All animals were observed twice daily for signs of morbidity or mortality. Clinical signs were recorded monthly. Body weights by cage were recorded every week for the first 12 weeks and monthly thereafter. The mean body weight of each group was calculated by dividing the total weight of all animals in the group by the number of surviving animals in the group. Moribund animals and animals that survived to the end of the study were killed with carbon dioxide and necropsies were performed. Examinations for grossly visible lesions were performed on major tissues or organs. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following were examined microscopically: tissue masses, abnormal lymph nodes, skin, mandibular or mesenteric lymph nodes, mammary gland, salivary gland, sternebrae, bone marrow, thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, small intestine, colon, liver, gallbladder (mice), pancreas, spleen, kidneys, adrenals, urinary bladder, prostate/ testes or ovaries/uterus, brain, and pituitary. Necropsies were performed on all animals unless precluded in whole or in part by autolysis or cannibalization.
The classification of neoplastic nodules in the liver was done according to the recommendations of Squire and Levitt (1975) and the National Academy of Sciences (1980).
When the pathology examination was completed, the slides, individual animal data records, and summary tables were sent to an independent quality assurance laboratory. Individual animal records and tables were compared for accuracy, slides and tissues were verified, and histotechniques were evaluated. All tumor diagnoses, target tissues, and tissues from a randomly selected 10% of the animals were evaluated by a pathologist. Slides of all target tissues, neoplasms, and other slides about which the original and quality assurance pathologists disagreed were submitted to the Chairperson of the Pathology Working Group (PWG) for evaluation. Representative slides selected by the Chairperson were reviewed blindly by PWG pathologists, who reached a consensus and compared their findings with the original diagnoses. When disagreements were found, the PWG sent the appropriate slides and their comments to the original pathologist for review. The final diagnosis represents a consensus of contractor pathologists and the NTP Pathology Working Group.
Data on this experiment were recorded in the Carcinogenesis Bioassay Data System (Linhart et al., 1974). The data elements include descriptive information on the chemicals, animals, experimental design, clinical observations, survival, body weight, and individual pathologic results, as recommended by the lnternational Union Against Cancer (Berenblum, 1969).
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
m-bis(2,3-epoxypropoxy)benzene
EC Number:
202-987-5
EC Name:
m-bis(2,3-epoxypropoxy)benzene
Cas Number:
101-90-6
Molecular formula:
C12H14O4
IUPAC Name:
m-bis(2,3-epoxypropoxy)benzene
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Ciba-Geigy Corporation (Ardsley, NY), Araldite ERE 1359 in a single lot (No. P-60002)
- Purity test date:
81.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
in the dark at 23 °C in its original container
- Stability under test conditions:
stable

FORM AS APPLIED IN THE TEST: suspended in corn oil (dissolved in acetone before being added to corn oil)

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
Charles River Breeding Laboratories, Portage, MI
- Age at study initiation:
8 to 9 weeks
- Weight at study initiation:
NA
- Fasting period before study:
NA
- Housing:
five animals/cage
- Diet:
ad libitum
- Water:
ad libitum
- Acclimation period:
3 weeks

DETAILS OF FOOD AND WATER QUALITY:
Feed: Wayne Laboratory Blox (r), Allied Mills (Chicago, IL)
Water: Edstrom automatic watering system, Edstrom Industries (Waterford, WI)

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
17-32
- Humidity (%):
20-81
- Air changes (per hr):
12 changes of room air per hour
- Photoperiod (hrs dark / hrs light):
12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Using the observations and results from the 13-week study, groups of 50 rats of each sex were administered diglycidyl resorcinol ether in corn oil by gavage 5 days per week for 103 weeks at doses of 0,25, or 50 mg/ kg body weight. Because of early deaths in the high dose male and female rats, a supplemental study using DGRE dose levels of 0 and 12 mg/ kg was started 12 months after the primary study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
One-milliliter aliquots of the sample vials were extracted with 10 mL of methanol containing 0.7 g/mL of dibutyl phthalate as an internal standard. A reference calibration plot was prepared from spiked samples which were extracted in the same manner. The supernatant solutions were analyzed by VPC-FID at 210° on a 6 ft x 1/4 in x 2 mm ID glass column packed with SP2250 on 100/120 Supelcoport.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
Daily, 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
First and Second Studies
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
First Study
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
First Study
Dose / conc.:
12 mg/kg bw/day (actual dose received)
Remarks:
Second study
No. of animals per sex per dose:
50 Males and 50 Males
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 13-week study observation and results
- Rationale for animal assignment (if not random): random

Examinations

Observations and examinations performed and frequency:
All animals were observed twice daily for signs of morbidity or mortality. Clinical signs were recorded monthly. Body weights by cage were recorded every week for the first 12 weeks and monthly thereafter. The mean body weight of each group was calculated by dividing the total weight of all animals in the group by the number of surviving animals in the group. Moribund animals and animals that survived to the end of the study were killed with carbon dioxide and necropsies were performed. Examinations for grossly visible lesions were performed on major tissues or organs. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following were examined microscopically: tissue masses, abnormal lymph nodes, skin, mandibular or mesenteric lymph nodes, mammary gland, salivary gland, sternebrae, bone marrow, thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, small intestine, colon, liver, gallbladder (mice), pancreas, spleen, kidneys, adrenals, urinary bladder, prostate/ testes or ovaries/ uterus, brain, and pituitary.
Sacrifice and pathology:
Necropsies were performed on all animals unless precluded in whole or in part by autolysis or cannibalization. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and is not necessarily equal to the number of animals that were placed on study in each group. The classification of neoplastic nodules in the liver was done according to the recommendations of Squire and Levitt (1975) and the National Academy of Sciences (1980). When the pathology examination was completed, the slides, individual animal data records, and summary tables were sent to an independent quality assurance laboratory. Individual animal records and tables were compared for accuracy, slides and tissues were verified, and histotechniques were evaluated. All tumor diagnoses, target tissues, and tissues from a randomly selected 10% of the animals were evaluated by a pathologist. Slides of all target tissues, neoplasms, and other slides about which the original and quality assurance pathologists disagreed were submitted to the Chairperson of the Pathology Working Group (PWG) for evaluation. Representative slides selected by the Chairperson were reviewed blindly by PWG pathologists, who reached a consensus and compared their findings with the original diagnoses. When disagreements were found, the PWG sent the appropriate slides and their comments to the original pathologist for review. (This procedure has been described by Maronpot and Boorman, 1982.) The final diagnosis represents a consensus of contractor pathologists and the NTP Pathology Working Group.
Statistics:
Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958).
Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not statistically censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone’s (1975) extensions of Cox’s methods for testing for a dose-related trend. All reported P values for the survival analyses are two-sided.
For the statistical analysis of tumor incidence data, two different methods of adjusting for intercurrent mortality were employed. Each used the classical method for combining contingency tables developed by Mantel and Haenszel (1959). Tests of significance included pairwise comparisons of high and low dose groups with controls and tests for overall dose-response trends.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Throughout most of the primary study (after week 30), mean body weights of high dose rats of each sex were lower than those of the controls. Except for weeks 80 to 100, mean body weights of low dose males and females were comparable with those of the con- trols. Wheezing and respiratory distress were the only compound-related clinical signs observed. not affected by the administration of 12 mg/ kg Body weight gain in the supplemental study was of DGRE.
Mortality:
mortality observed, treatment-related
Description (incidence):
The survival of male and female rats was significantly reduced (P
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Lung: Bronchopneumonia was the most frequent cause of early death in rats and was characterised by patches of polymorphonuclear leukocytes in the alveoli of the lung, especially near the bronchi. Polymorphonuclear leukocytes also occurred in masses within bronchi and in the bronchial epithelium. In some rats, pulmonary vessels were dilated and showed perivascular edema. Microbiological examinations were not conducted on these rats. Pneumonia was not observed in the supplemental study.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach: Diglycidyl resorcinol ether produced hyperkeratosis, hyperplasia, and neoplasms of the squamous epithelium of the forestomach in both the primary and the supple mental studies (Tables 8 and 9). The squamous epithelium of the esophagus and nasopharynx was hyperkeratotic in some rats, but no tumors were found.

Postmortem examination of the stomachs revealed numerous small rough nodules on the nonglandular mucosa that progressed in some animals to form large, white, fungiform masses which were occasionally ulcerated. The larger lesions involved adjacent tissues such as the spleen, pancreas, and lymph nodes. Histologically, the thickened mucosa showed intense hyperkeratosis that was usually accompanied by hyperplasia of the basal layers. Small nodules, diagnosed as squamous papillomas, were characterized by projections of hyperkeratotic epithelium supported by a fibrovascular core. These changes appeared to be identical to those found in the 13-week study.

In the larger masses, the basal cells developed hyperchromatism, pleomorphism, and parachromatin clearing all signs of malignancy. The masses grew downward through the basement membrane into and through the muscularis mucosa in irregular strands and clumps, and keratin pearls were often produced. Both normal and abnormal mitotic figures were frequently observed. These lesions were diagnosed as squamous cell carcinomas. Metastases from these tumors were found in 14 low dose males, 1 high dose male, and 5 low dose females. Metastatic tumors were found in the regional lymph nodes, pancreas, liver, spleen, lungs, and brain.

Low and high dose male and female rats had statistically significant increased incidences of squamous cell papillomas and carcinomas, although the effects in the high dose groups were not as striking (Tab1es 10 and 1I). The markedly increased number of early deaths in high dose male and female groups may explain the low incidences of benign and malignant neoplasms of the nonglandular stomach at this dose level. The supplemental study (1 2 mg/ kg) also showed an extremely high rate of benign (males: 32%; females:, 38%) and malignant (males: 78%; females: 54%) neoplasms in the forestomach. No benign or malignant neoplasms were observed in the control rats of either sex.

Other Sites: In the primary study, several other types of neoplasms occurred in rats with overall incidences that were lower in the dosed groups than in the controls; these included adrenal pheochromocytoma, leukemia, pituitary adenoma, and thyroid C-cell tumors in males and females; lung adenoma, pancreatic islet cell tumors, and interstitial cell tumors of the testes in males; and mammary gland fibroadenomas and uterine tumors in females. None of these decreases were statistically significant when life table analyses were used, and they appeared to be related to the reduced survival observed in the dosed groups relative to those in the controls. In the supplemental study, neurofibrosarcomas were observed at an increased incidence in dosed male rats (control, O/50; dosed, 31 50), but the increase was not statistically significant. The incidence of C-cell tumors of the thyroid was significantly reduced in the dosed males compared to controls (control, 11/50; dosed, 3/50; P<0.03, incidental tumor and Fisher exact tests). In female rats, there was a statistically significant decrease (P<0.05) in the incidence of pheochromocytomas of the adrenal medulla in the dosed group (control, 5/50; dosed, Oj50).
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
LOAEL
Effect level:
12 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
12 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, technical grade diglycidyl resorcinol ether (DGRE) caused hyperkeratosis and hyperplasia of the forestomach in rats. DGRE was carcinogenic for male and female F344/N rats, causing both benign and malingnant neoplasms of the forestomach.
Under the conditions of the study, the LOAEL may be assigned to 12 mg/kg bw/day dose. It is however not excluded, that the real LOAEL value may be lower as under given testing conditions. On the other hand, the chronic toxicity of DGRE via oral exposure is not relevant to humans. There was also an opinion, that the observed forestomach tumors were likely to have resulted from an indirect or local toxic effect of DGRE. Therefore by assessing the substance hazard for humans it is essential to identify the effects of toxicity from the other than oral exposure routes.
Executive summary:

Toxicology and carcinogenesis study of technical grade diglycidyl resorcinol ether (81% pure) was conducted by administering the chemical in corn oil by gavage to groups of 50 male and 50 female F344/N rats at doses of 25 or 50 mg/kg. A supplemental study of similar design in male and female rats (0 or 12 mg/kg)was started approximately 12 months later because of high mortality in the 50 mg/kg dose groups. Doses were administered five times per week for 103 weeks. Groups of 50 rats of each sex received corn oil by gavage on the same dosing schedule and served as vehicle controls.

Throughout most of the primary study, mean body weights of high dose male and female rats were lower than those of the corresponding vehicle controls. In the supplemental study, body weights of both sexes of the dosed rats were unaffected by administration of DGRE. Survival of dosed rats of each sex in the primary study was dose related and was shorter (P<0.001) than that of the vehicle controls. No high dose male rats and only 1/50 high dose female rats lived to the end of the study. Bronchopneumonia was the most frequent cause of early death among the rats and may have resulted from the animals’ aspiration of corn oil containing diglycidyl resorcinol ether. Survival of the dosed male rats in the supplemental study was reduced (P<0.005) when compared to controls. There was no significant difference in survival between dosed and control female rats in the supplemental study.

The incidences of rats with hyperkeratosis and hyperplasia of the forestomach were compound related. For rats of each sex, incidences of animals with squamous cell papillomas, squamous cell carcinomas, or both occurred with statistically significant positive trends and the incidences in the dosed groups were significantly higher than those in the vehicle controls.

The significantly lower survival of rats in the high dose groups probably reduced the incidence of stomach neoplasms in these groups and was responsible for the numerous decreased overall tumor incidences observed in other organs in dosed groups relative to the controls.

An audit of the experimental data was conducted for the 2-year studies of diglycidyl resorcinol ether. No data discrepancies were found that influenced the final interpretations.

The authors concluded, that under the condition of the 2-year gavage study, technical grade diglycidyl resorcinol ether caused hyperkeratosis and hyperplasia of the forestomach in rats. DGRE was carcinogenic for male and female F344/N rats, causing both benign and malignant neoplasms of the forestomach.

Under the conditions of the study, the LOAEL may be assigned to 12 mg/kg bw/day dose. It is however not excluded, that the real LOAEL value may be lower as under given testing conditions. On the other hand, the chronic toxicity of DGRE via oral exposure is not relevant to humans. There was also an opinion, that the observed forestomach tumors were likely to have resulted from an indirect or local toxic effect of DGRE. Therefore by assessing the substance hazard for humans is essential to identify the effects of toxicity from the other than oral exposure routes.

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