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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 May 1989 - 3 August 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OPTS Subchronic Oral Toxicity Studies, 40 CFR, Part 796.2650 and under GLP. No details on test substance compostion, responsabilty of the sponsor.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Compared to the current OECD 408 guideline.

- Deviations from the desired temperature and humidity ranges.
- No substance identification data besides lot number and % main consituent.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FORMAL [BIS(2-Chloroethoxy)Methane]
- Physical state: Clear liquid
- Analytical purity: Approximately 85% active ingredient. The identity, strength, purity and composition; and synthesis, fabrication, and/or derivation of the test substance have been documented by the sponsor.
- Impurities (identity and concentrations): Documented by the sponsor.
- Composition of test material, percentage of components: Documented by the sponsor.
- Lot/batch No.: 237-81
- Expiration date of the lot/batch: not indicated
- Stability under test conditions: The stability of the test substance has been determined by the sponsor.
- Storage condition of test material: At room temperature in a temperature monitored room.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding laboratories, Inc., Kingston, New York 12484
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: Males 189 (174-203), females 147 (138-156)
- Fasting period before study: Not applicble
- Housing: two per cage in elevated stainless steel wire mesh cages during the first week of the acclimation period and individually housed thereafter.
- Diet (e.g. ad libitum): Ad libitum; standard laboratory diet (Purina Certified Rodent Chow Brand Animal Diet #5002). Fresh food presented weekly.
- Water (e.g. ad libitum): Ad libitum; by automated watering system (Elizabeth town Water Company)
- Acclimation period: Animals were acclimated for 2 weeks (17 April 1989 to 1 May 1989). All animals were examined by the staff veterinarian during the acclimation period .

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 18-66
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: 2 May 1989 - 3 August 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of the test substance were diluted in the vehicle weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 100 mg/ml
- Amount of vehicle (if gavage):
dose group
1 = 1.2 ml/kg
2 = 0.1 ml/kg
3 = 0.2 ml/kg
4 = 0.4 ml/kg
5 = 0.8 ml/kg
6 = 1.2 ml/kg
- Lot/batch no. (if required): Buy before 5 Aug 5 89B
- Purity: Not appicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of dosing mixtures for homogeneity, stability and verification of concentration here performed by the sponsor. Samples were obtained as
follows and submitted for analysis to Dr. Harren Duncan, Morton International, Inc., 1275 Lake Avenue, Woodstock, Illinois 60098:
Pretest homogeneity - 2 samples, one each from the top and bottom (28 April 1989).
Confination of concentration - one sample from the dose mixtures at weeks 4, 8, and 14 (8 and 22 May 1989, 19 June 1989 and 9 August 1989,
respectively) .
Duration of treatment / exposure:
Three months
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 20, 40, 80, 120 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range finding study
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): No data
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Pre-test and weekly thereafter

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Twice pretest, weekly during treatment and terminally

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: Weekly beginning one week prior to treatment in g/kg/day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and a study termination
- Dose groups that were examined: all
HAEMATOLOGY: Yes
Blood was obtained via venipuncture of the orbital sinus under light ether anesthesia.
- Time schedule for collection of blood: Performed pretest on 10 aninals/sex (not placed on study) and on all surviving animals at Month 1 and study ternination.
- Anaesthetic used for blood collection: Yes (identity) ether
- Animals fasted: Yes
- How many animals: Performed pretest on 10 aninals/sex (not placed on study) and on all surviving animals at Month 1 and study ternination.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
Blood was obtained via venipuncture of the orbital sinus under light ether anesthesia.
- Time schedule for collection of blood: Performed pretest on 10 aninals/sex (not placed on study) and on all surviving animals at Month 1 and study ternination.
- Animals fasted: Yes
- How many animals: Performed pretest on 10 aninals/sex (not placed on study) and on all surviving animals at Month 1 and study ternination.
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Complete gross postmortem examinations were performed on all animals. External surface, all orifices, the cranial cavity, the external surfaces of the
brain and spinal cord, nasal cavity , the thoracic, abdominal and pelvic cavities with their associated tissues and organs and the neck with its associated tissues and organs were examined for all animals. Animals were fasted prior to scheduled sacrifice.

Organs Weighed and Organ/Body Weight and Organ/Brain Weight Ratios Calculated:
adrenals, brain, heart, kidneys, testes with epididymides, liver, ovaries

Tissues preserved:
adrenals (2), aorta (thoracic) bone (femur, including articular surface, and sternum), bone marrow (sternum), bone marrow smear (sternum), brain (medulla/pons , cerebral cortex, and cerebellar cortex), esophagus, eyes (2), heart, intestine, cecum, colon, duodenum, ileum, jejunum, rectum, kidneys (2), lacrimal gland, liver (2 sections), lungs (with mainstem bronchi - 2), lymph nodes (mesenteric and mediastinal), mammary gland (right inguinal w ith skin), nose (nasal tissues - 4 sections), ovaries (2), pancreas, pituitary gland, pharynx, prostate, salivary gland (mandibular), sciatic nerve, seminal vesicles, skeletal muscle (biceps femoris), skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach (glandular, nonglandular), testes with epididymides (2), thymus, thyroid/parathyroids (2), trachea, urinary bladder, uterus, Zymbal glands, gross lesions

HISTOPATHOLOGY: Yes
All tissues listed above were examined for all animals in Groups I and VI (control and high-dose groups, respectively) and for all animals dying prior to terminal sacrifice. The kidneys, liver, lungs and all gross lesions were examined for all animals in the intermediate-dose groups (Groups I I , I I I, IV and V) .
Statistics:
Body weights, food consumption, hematology and clinical chemistry parameters, terminal organ weights and organ/body and organ/brain weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All ten males and seven of the ten females receiving the highest dose of the test substance (120 mg/kg/day; Group VI) died or were killed in a
moribund condition during the study. Deaths occurred as late as Day 76. One female in the group receiving 80 mg/kg bw/day (Group V) was found
dead on day 78 of the study. No substance related mortality attributable to FORMAL administration occurred at lower dose levels (10, 20, and 40 mg/kg/day).
No cause of death was apparent from gross postmortem examinations, however, microscopic examination revealed degeneration of the myocardium, considered to be a possible cause of death, in all animals which died after Day 14.

BODY WEIGHT AND WEIGHT GAIN
Body weights of high-dose (120 mg/kg bw/day) males were markedly lower than weights of control males throughout the study, 17% in first week, 18% in second week and 7 to 21% in animals surviving after that. No statistically significant effecst in the other dose groups. No effecst observed in females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Males ta 120 mg/kg bw/day in week 1 and 2 were significantly lower then the control values. Food consumption of surviving animals and feamles was comparbale to the controls.
In the lower dose group the food consumption of the animals was higher, sometimes significantly, in the exposed group than in the control group. Although these differences are suggestive of a relationship to the substance administration, an increase in food consumption is not considered to represent an adverse or toxic effect.

CLINICAL CHEMISTRY
Some parameters were affected in the higher dose groups but the toxicclogical significance of these isolated differences, in the absence of other evidence of systemic toxicity, is considered questionable. No effect of test substance administration on clinical chemistry parameters was evident in Groups receiving 10, 20 or 40 mg/kg/day.

ORGAN WEIGHTS
Mean liver weights, liver/body weight ratios and liver/brain weight ratios for males and females receiving doses of 40 or 80 mg/kg/day and for
females receiving 120 mg/kg/day were higher than control values at study termination. Differences for the two highest dose groqx of females (80 and 120 mg/kg/day) were statistically significant (p <0.01). Microscopic pathology examinations revealed hypertrophy of central lobular hepatocytes, which would be consistent with increased liver weights, in males in Groups IV and V (40 and 80 mg/kg/day) but this observation was not seen in treated females.
Mean adrenal weights, both absolute and relative to body and brain weight, for males receiving 20, 40 and 80 mg/kg/day were slightly lower than the
mean control weight; differences were statistically significant for one or more of the indices. Similar differences were not clearly evident in females and no morphologic abnormalities were seen in adrenal s examined microscopical ly. The toxicological significance, if any, of these differences is not clear.
The few other statistically significant differences noted (elevated testes/body weight ratio and kidneylbody weight ratio for males receiving 80 mglkglday) appear to reflect the relatively low body weights in this group and were not considered indicative of test substance-related toxicity.


GROSS PATHOLOGY/HISTOPATHOLOGY: NON-NEOPLASTIC
Gross postmortem examinations confirmed the emaciation and/or poor condition seen in a few high-dose (120 mg/kg bw/day) animals during the antemortem phase of the study. No cause of death was evident from gross postmortem examinations of animals from Groups V and VI (80 and 120 mg/kg bw/day) which died during the study; however, microscopic examination revealed myocardial degeneration, considered to be a possible cause of death, in all animals in this group which died after Day 14.
Other morphologic abnormalities which appeared to be related to the test substance were seen in the liver, kidneys, thymus, spleen, bone marrow,
brain and spinal cord.

Heart:
Myocardial degeneration, slight to moderate in severity, was seen in several high-dose males and females and in the one female from the 80 mg/kg bw/day group for which the heart was examined microscopically (the animal which was found dead). This finding tended to be somewhat more pronounced in males than in females. Characteristic features included cytoplasmic swelling, decreased intensity of staining characteristics, loss of cross striations and vacuolization and nuclear pyknosis of the myocardial fibers accompanied by an apparent increased cellularity of the interstitium. Myocardial degeneration was seen primarily in animals which died on or after the second week of study.

Liver:
Hypertrophy of central lobular hepatocytes, minimal to slight in severity, was seen in a number of treated males, but not females, from groups receiving 20, 40 and 80 mg/kg/day (Groups III, IV and V). Based on both incidence and severity, this was most pronounced in Group V. These males were all
animals which survived to study termination. The fact that this finding was not apparent in males receiving 120 mg/kg bw/lday (Group VI) , all of which died prior to the end of the study, suggests that this may be an effect which requires a longer treatment interval than that which was received by Group VI males in this study. (The longest-surviving animal in this group died on Day 66.)

Kidneys:
Minimal to moderate tubular nephrosis was seen in one or more males from all groups. A number of these animals had birefringent intracytoplasmic
inclusions in the epithelial cells lining the affected convoluted tubules. Both findings were dose-related, i .e., most pronounced in Group V (80 mg/kg bw/day) followed by Groups IV and III (40 and 20 mg/kg bw/day) . In groups I an II (10 mg/kg/day) findings were essentially comparable. Intracytoplasmic hyaline droplets in the proximal convoluted tubules were also seen in numerous males from all groups, but this was more pronounced in animals in Groups IV and V than in the control animals and animals in Groups II and III. These findings also appear to represent a longer-term effect since they were seen only in high-dose (Group VI) males which died after the second week on test.

Thymus, Spleen and Bone Marrow:
A sl ight increase in incidence and severity of involution/atrophy and
lymphocytolysis/necrosis of the thymus was evident in high-dose (Group VI) animals when compared to incdence and severity of this observation in control animals. Slight to moderate splenic atrophy and moderate to marked hypcellularity of the sternal and femoral bone marrow were also seen in a small number (one to three per sex) of males and females from this group. Similar findings were not seen in any control animals. Although these findings are suggestive of an effect of FORMAL administration, heir toxicological significance, if any, is considered equivocal.

Brain and Spinal Cord:
Vacuolization and gliosis were seen in the brain stem of a small number of males and femles (one or two per sex) from Group VI; both findings were minimal to slight in severity. In the affected males, minimal to moderate vacuolization and gliosis involving the grey matter of the cervical and thoracic portions of the spinal cord were also seen. Cerebellar malacia and edema (of moderate severity) were also seen in one female. Because these morphological abnormalities in the brain and spinal cord , while unusual, were seen in on small number of animals, their toxicological significance is not clear.

Other findings were not considered to be related to administration of the test substance.

Effect levels

Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Based on microscopic evidence of renal and hepatic pathology in males receiving 20 mg/kg/day, the no observed effect level (NOEL) for FORMAL when administered orally to rats for three months under conditions of this study was 10 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analysis of Dosing Mixtures:

Analyses, performed by the sponsor, confirmed that the procedure used to prepare the mixture produced homogeneous mixtures which were of the appropriate concentration and were stable for at least three months after Preparation. Pretest homogeneity analysis demonstrated concentrations at the top and the bottom of the mixtures which were 103% and 102% of the nominal (desired) concentration. Analysis of mixtures prepared at Week 1, 4, 8 and 13 demonstrated concentrations of 101, 99, 97, and 108% of the nominal concentration. A sample of the mixture prepared for Week 1 was analyzed after 13 weeks and shown to contain 99.7% of the nominal concentration, thus demonstrating stability of the test substance in corn oil over this interval.

Applicant's summary and conclusion

Conclusions:
Based on microscopic evidence of renal and hepatic pathology in males receiving 20 mg/kg/day, the no observed effect level (NOEL) for FORMAL when administered orally to rats for three months under conditions of this study was 10 mg/kg/day.
Executive summary:

This study, conducted for Morton International, Inc., was designed to assess the potential subchronic toxicity of FORMAL [BIS (2-Chloroethoxy) Methane] when administered orally, via gavage to rats. This study was designed to follow the TSCA (Toxic Substances Control Act) U.S. Environmental Protection Agency, Off ice of Pesticide and Toxic Substances, Subchronic Oral Toxicity Studies, 40 CFR, Part 796.2650. This study was conducted in compliance with Part 792 of 40 CFR (EPA/TSCA Good Laboratory Practice Regulations). The test substance, diluted in corn oil at a concentration of 100 mg per ml was administered to 100 Sprague-Dawley CD rats (10/sex/group) at dose levels of 10, 20, 40, 80 and 120 mg/kg/day for a period of at least 90 days. Control animals (10/sex) received the vehicle (corn oil) at the same dose volume as administered to the high-dose group. Physical observations and body weight and food consumption measurements were performed for all animals pretest and weekly during the study period. Ophthalmoscopic examinations were performed for all animals pretest and at study termination. Hematology and clinical chemistry evaluations were performed on 10 animals/sex pretest (animals not used in the study) and on all surviving animals at month 1 and at study termination. After at least 3 months of treatment, all survivors were sacrificed, selected organs were weighed and terminal organ/body and organ/brain weight ratios calculated. Complete gross postmortem examinations and histopathological evaluation of all gross lesions were conducted for all animals. Histopathological evaluation of all preserved tissues was conducted on all animals in Groups I and VI (control and high-dose groups, respectively) and on all animals dying prior to study termination. The lungs, liver, and kidneys were examined histopathologically for all animals in the intermediate-dose groups (Groups II, III, IV, and V ) . Analyses of dosing mixtures confirmed that the procedure used to prepare the mixture produced homogeneous mixtures which were of the appropriate concentration and were stable for at least three months after preparation.

All ten males and seven of the ten females receiving the highest dose of the test substance (120 mg/kg/day) died or were killed in a moribund condition during the study. The first death occurred after a single dose and seven deaths occurred during the first week of the study. The mortality rate declined subsequently, but additional deaths occurred as late as Day 76. No cause of death was apparent from gross postmortem examinations; however, microscopic examination revealed degeneration of the myocardium, considered to be a possible cause of death, in all animals which died after Day 14, One female in the group receiving 80 mg/kg/day was found dead on Day 78 of the study. Mortality in animals receiving 80 or 120 mg/kg/day was considered to be related to test substance administration. No mortalitv attributable to FORMAL administration occurred at lower dose level s (10, 20 - and 40 mg/kg/day) .

Antemortern abnormalities seen in animals which were killed in a moribund condition and a few of the animals which were found dead, included emaciation, poor food consumption, general poor condition, hypothermia, lethargy/prostration, dypsnea/gasping, moist rales, ataxia, abnormal posture, slight tremors, salivation, and brown/yellow stains on the snout, paws, surface and/or anogenital area. No effect of test substance admini stratian was evident in physical observations of animals which survived. Ophthalmoscopic examinations at study termination revealed no ocular abnormalities attributable to Formal. Body weights of high-dose (120 mg/kg/day) males were markedly lower than weights of control males throughout the study. The mean weights for the six animals in this group surviving at the end of the first week and the four animals surviviving at the end of the second week were 17 and 18% lower than the concurrent control means. Subsequent differences from the mean control weight for the one or two surviving animals ranged from 7 to 21%. Mean weights of males receiving 80 mg/kg/day were slightly lower than control weights during the second two months of the study; differences ranged from 6% at Week 5 to 10% at Week 12 and are considered suggestive of an effect of test substance administration. Body weights for males in the 10, 20 and 40 mg/kg/day groups were considered comparable to control weights. No effect on body weight was evident in females at any dose level. on ocbund ation, ture, ventral Mean food consumption values for high-dose (120 mg/kg/day) males at Weeks 1 and 2 were lower than mean control values; this is consistent with the mortality and low body weight gains in this group. No other food consumption differences considered to represent an adverse or toxic effect of Formal administration were seen.

No effect of FORMAL administration on hematology or clotting parameters was apparent. Alterations of clinical chemistry parameters considered to be indicative of an effect of FORMAL administration at the high dose level (120 mg/kg/day ) consisted of elevations in serum glutamic oxoal acetic transaminase (SGOT) and alkaline phosphatase levels for both sexes and elevations in blood urea nitrogen (BUN) values for females. SG0T values were slightly elevated relative to control values in both sexes at Month 1 and markedly elevated in females at study termination. The serum alkaline phosphatase value for high-dose males was statistically significantly higher than the control value at Month One. Alkaline phosphatase and BUN values for high-dose females were slightly higher than control ialues at Months 1 and 3. No clear effect of test substance administration on clinical chemistry parameters was evident in Groups receiving 10, 20, 30 or 80 mg/kg/day. Mean liver weights, liver/body weight ratios and liver/brain weight ratios for males and females receiving doses of 40 or 80 mg/kg/day and for females receiving 120 mg/kg/day were higher than control values at study termination. Microscopic pathology examinations revealed hypertrophy of central lobular hepatocytes, which would be consistent with increased liver weights, in males in Groups IV and V (40 and 80 mg/kg/day) but this observation was not seen in treated females. Mean adrenal weights, both absolute and relative to body and brain weight, for males receiving 20, 40 and 80 mg/kg/day were slightly lower than the mean control weight. Similar differences were not clearly evident in females and no morphologic abnormalities were seen in adrenals examined microscopically. The toxicological significance, if any, of these differences in adrenal weights is not clear. Gross and microscopic postmortem examinations revealed changes in the heart, liver, kidneys, thymur , spleen, bone marrow, brain and spinal cord which appeared to related to test substance administration, Changes in the heart consisted of moderate to severe myocardial degeneration which was seen in all high dose animals (120 mg/kg/day) which were found dead or killed in a moribund condition after Day 14 and in the one female in the 80 mg/kg/day group which was found dead. This was considered to be a possible cause of death in these animals. Dose-related microscopic alterations in the liver and kidney were evident in males (but not in females) receiving 20, 40 or 80 mg/kg/day. Liver pathology was characterized by minimal to slight hypertrophy of the central lobular hepatocytes. The fact that this finding was not apparent in males receiving 120 mg/kg/day, all of which died prior to the end of the study, suggest that this may be an effect which requires a longer treatment interval than that which was received by these animals. Kidney changes included an increased incidence of minimal to moderate tubular nephrosis with bi refringent intracytoplasmic inclusions in the convoluted tubular epithelium. An increased incidence and severity of intracytoplasmic hyaline droplets in the proximal convoluted tubules was also evident in males in the 40 and 80 mg/kg/day groups. Similar findings (renal changes) in the 120 mg/kg/day group were seen only in males which died after the second week of test, suggesting that these findings also represent a longer-term effect.

Several additional microscopic alterations seen in other organs of animals from the high-dose groups were considered suggestive of an effect of test substance administration. (These organs were not examined for animals in the other dose groups.) Unusual morphologic abnormalities of the brain and spinal cord which were seen in the high-dose animals only (not in controls) included vacuolization and gliosis in the brain stem (a few males and females) and the cervical and thoracic spinal cord (males only). Moderate cerebellar malacia and edema were also seen in one female. Changes in the spleen and bone marrow which occurred in high-dose animals and were not seen in control animals consisted of slight to moderate splenic atrophy and moderate to marked hypocel lularity of the sternal and femoral bone marrow in a few animals (one to three per sex). A slight increase in incidence and severity of involution/atrophy and lymphocytolysis/necrosis of the thymus was also evident in high-dose animals when compared to incidence and severity of this observation in control animals. Based on microscopic evidence of renal and hepatic pathology in males receiving 20 mg/kg/day, the no observed effect level (NOEL) for FORMAL when administered orally to rats for three months under conditions of this study was 10 mg/kg/day.