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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:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for Testing of Chemicals, No. 451, May 12, 1981
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-iminodi(ethylamine)
EC Number:
203-865-4
EC Name:
2,2'-iminodi(ethylamine)
Cas Number:
111-40-0
Molecular formula:
C4H13N3
IUPAC Name:
N-(2-aminoethyl)ethane-1,2-diamine
Details on test material:
Dihydrochloride salt in DETA

The dihydrochloride salt of DETA (diethylenetriamine), produced by reacting diethylenetriamine with commercial concentrated hydrochloric acid in a solvent mixture of ethanol and toluene, was used as the test material (CAS Registry # 3488-90-2). Three 1-gallon containers and one 1-quart container of the test material were received from the Union Carbide Corporation (south Charleston, WV) on September 15, 1986 and were identified with the lot number 36-ARD-24. The material was assigned the BRRC Numbers 49-346-A, 49-346-B, 49-346-C, and 49-346-D. The purity of the test material was greater than 99% and the test material was stable through the course of the study as determined by the Union Carbide Corporation (s. Charleston, WV). The purity and stability analyses of the test material measurements were based on the weight of the dihydrochloride salt of diethylenetriamine, and no corrections were made for the HCl component. This approach was used since the dose levels were established based on results from a study that used the salt form of the test material. The test material was an off-white to tan crystalline material and was stored at room temperature.

Test animals

Species:
rat
Strain:
other:
Sex:
male/female
Details on test animals or test system and environmental conditions:
Two hundred and sixty-two Fischer 344 rats (130 males and 132 females) were purchased from Charles River Breeding Laboratories, Inc. (Kingston, NY). The females were nulliparous and non-pregnant. The animals were received on March 2, 1986 and were approximately 28 days of age. The rats were approximately 6 weeks of age at first dose.

One day after arrival, blood was collected from 5 animals/sex for serologic virology titer determinations, and five animalsfsex were examined
for fecal parasites. Parasite examination consisted of the cellophane tape test of perineal skin and hair for pinworms and a zinc sulfate flotation test on the cecal and/or colon contents. In addition, 3 animals/sex were subjected to histologic examination of at least the salivary glands, trachea, lungs, liver, kidneys, spleen,heart, submandibular lymph nodes, and nasal cavities.

The rats were housed one animal per side of divided stainless steel cages (solid sides with wire mesh floors) mounted in a stainless steel Maxi-Rack@ (Hazleton Systems, Inc., Aberdeen, MD). A layer of Deotized Animal Cage Board@ (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was kept under each cage and changed at least three times per week. Other animal care procedures were performed regularly according to BRRC Standard Operating Procedures. Water was available ad libitum. The water (Municipal Authority of Westmoreland County, Greensburg, PA) was administered by an automatic watering system with demand control valves mounted on each rack. Ground Purina Certified Rodent chow@ 55002 (Ralston Purina Co., St. Louis, MO) was available ad libitum. Room temperature and relative humidity were monitored continuously. Temperature was routinely maintained between 66" and 77'F, relative humidity between 40 and 70%. A 12-hour light/dark cycle each day was used (0500 to 1700 light).

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Experimental diets were prepared once each week using the following procedure. The dihydrochloride salt of DETA was ground with a Karzzeitbetrieb grinder and added to ground chow to prepare a premix at a concentration of 30000 ppm and mixed for 30 minutes. The high dose (15,000 ppm) diet was prepared by dilution of this premix and mixed for 45 minutes. The mid and low dose group preparations were prepared by dilution of the high dose diet with the appropriate amount of ground chow and mixed for 15 minutes. Fresh diets were offered to the animals each week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Experimental diets were analyzed using a high pressure liquid chromatography procedure developed at BRRC. Homogeneity and stability of the test material under the storage conditions employed in the study for each diet concentration were established prior to the start of the study. Diet concentrations were verified for all dose levels for the first four weeks of the study prior to administration of the diets to the animals. In subsequent weeks, a sample of one of the test diets (selected sequentially) was retained from each week's preparation and analyzed with one control sample in weeks 8 and 13.
Duration of treatment / exposure:
90 Days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000, 7500, or 15000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
70, 530, 1060 mg/kg bw/day and 80, 620, 1210 mg/kg.day for males and females, respectively
Basis:
actual ingested
No. of animals per sex per dose:
Twenty- 1000 ppm and 7500 ppm
Thirty - control and 15,000 ppm
Control animals:
yes
Details on study design:
The additional ten animals per sex included in the 0 and 15000 ppm dose groups were used for a post-exposure recovery phase during which control feed was administered to all animals.

During the 13-week exposure period, observations for mortality were made twice daily (a.m. and p.m.). Detailed clinical observations were performed
once each week, and observations for overt clinical signs were made on all other days. Body weight and food consumption data were collected for all
animals weekly. Ophthalmic examinations were performed, using an indirect ophthalmoscope, prior to dosing and prior to final sacrifice. Prior to
fasting for the bleeding procedures, urine was collected for 24 hours from 10 animals/sex/group in weeks 6 and 13. Blood was obtained prior to dosing (10 animals/sex), and in the 6th and 13th weeks of the study (10 animals/sex/ group) from methoxyflurane anesthetized rats via the retroorbital sinus for clinical chemistry and hematology analyses. Supplemental samples were obtained from untreated animals (culls) 1 day after the start of dosing for pre-study clinical chemistry and hematology analysis due to insufficient blood volumes from the original 10 animals/sex/group. Following the 13-week bleeding, the animals were sacrificed as described below.

Following 13 weeks of exposure, the remaining animals from the control and high dose (15000 ppm) groups were monitored for four weeks. Body weight and food consumption data were collected weekly during the recovery phase of the study. Observations were performed as described above. Following the 4-week recovery period (i.e. during week 171, urine was collected for 24 hours 1-2 days prior to sacrifice. Blood was collected just prior to sacrifice from all animals (fasted) as described above for clinical chemistry and hematologyanalyses. Following bleeding, the animals were sacrificed.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
Post observation period: 4 weeks (control and high dose group only)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Pre-dose ophthalmic examinations
Statistics:
Data for continuous, parametric variables were intercompared for the dose and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate variance t-tests. Non-parametric data were analyzed by the Kruskal-Wallis test or by the Wilcoxon rank sum test as modified by Mann-Whitney. Frequency data were compared using Fisher's exact tests where appropriate. All statistical tests, except the frequency comparisons were performed using BMDP Statistical Software (Dixon, 1985). The frequency data tests are described in Biometry (Sokal, R. R. and Rohlf, F. J., W. H. Freeman and Company: San Francisco, 1969). The fiducial limit of 0.05 was used as the critical level of significance for all tests.

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:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Details on results:
Several treatment-related effects on in-life, clinical pathology and organ weight measurements were observed for the mid- and high dose groups through the 13-week treatment period. Decreases in food consumption in males and females at the high dose. Dose-related decreases in body weight or weight gain in mid- and high-dose groups. Increased MCV and MCH in males in the mid and high-dose groups. In females, decrease glucose and albumin, and increased MCV in the high-dose group, and similar changes in glucose and MCV in the mid-dose group. Dose-related increases in WBC and lymphocytes were found in mid- and high-dosed females. Increased urine pH in females and mid- and high dose groups, possibly associated with increase in kidney weight and/or excretion of test material was observed after 13-weeks of exposure. In females, increased kidney and liver weight (mid- and high dose), and adrenal (high dose only). During recovery, female rat food consumption was greater than the concurrent control group and body weights returned to control levels. Male body weights remained lower than the controls throughout the recovery period.

Analytical Chemistry
The range of the analyses for the homogeneity tests was 89.4 to 100.7% of nominal (Mean +/- SD = 95.1% +/-24.4) for the 1000 ppm diet, 91.0 to 109.5% of nominal (Mean +/- SD = 98.3% +/- 6.9) for the 7500 ppm diet, and 92.9 to 107.9% of nominal (Mean +/- SD= 98.2% +/- 4.9) for the 15000 ppm diet.

Stability studies were conducted on diets stored at room temperature in closed polyethylene containers (as stored during the study) and in open glass feeder jars. These analyses indicated that DETA remained stable for at least 7 days in open rat feeders, and for at least 21 days in closed polyethylene containers, both stored at room temperature. The values of analyses for the stability tests for all three diets ranged from 91.4 to 115.8% of nominal when assayed from 7 to 23 days after preparation. Concentration verification analyses of diets prepared for the study ranged from 90.6 to 108.6% of
nominal, and all diets were found to be acceptable for use.

Clinical Observations
No clinical signs related to treatment with DETA were observed in either sex at any dose level over the ninety-day exposure. The frequent observations of corneal opacity in all groups, including controls, are similar to observations that occur with regularity in this strain of rat from this vendor and were not considered related to treatment nor of consequence to evaluation of the toxicity of DETA.

Males
Food consumption in males from the high dose (15000 ppm) group tended to be slightly less than controls intermittently throughout the treatment period. Statistically significant reductions in food consumption compared to controls were observed for males in the high dose group for each of the first 4 weeks and in weeks 6 to 7, 9 to 10, and 12 to 13 (4 to 12% decrease). A statistically significant decrease in food consumption was also observed in the males at the mid dose (7500 ppm) in the first week of the study (-6%). No other effects on food consumption were observed in males at the 15000 or 7500 ppm dose levels. No effect on food consumption in males from the low dose group was observed. During the recovery phase (weeks 14-17), food consumption for the males from the treated group was similar to that of the control group.

Females
Food consumption for the females in the high dose (15000 ppm) group, as observed for the males of the high dose group, tended to be slightly lower than the controls with some intervals equivalent to the control group. Statistically significant reductions in food consumption compared to controls were observed for females in the high dose group only during the first 5 weeks of the study (approximately 6-9%). A statistically significant decrease in food consumption was noted in the females from the low dose group (approximately 7%) during study week 6. The statistically significant difference was considered spurious due to the lack of dose response, lack of effects during other measurement periods. No other effects on food consumption were observed in females at any dose level. During the recovery phase (weeks 14-17), food consumption was increased (10 to 16%) compared to the controls in the recovery group females.

Body Weights
Males
Body weight (5 to 7% decrease) and weight gain (12 to 29% decrease) were reduced compared to controls in the males from the high dose group (15000 ppm) throughout the study. Mean body weight for males from the 7500 ppm group was below that of the controls at each measurement period although these differences were not consistently statistically significant until the ninth week (3% decrease) of the study. Body weight gain in males from the mid dose group were reduced (4 to 12%) throughout the study. No treatment-related changes in male body weight or body weight gain at the 1000 ppm dose were observed during the study .

Body weights and body weight gains for the males in the recovery group showed only minimal recovery during the 4-week recovery period and remained lower than the control group through week 17.

Females
Females showed a similar pattern of absolute body weight and weight gain as the males during the treatment period. The statistical significance of mean body weight gains for the females in the 7500 ppm group was less consistent than that for the males, however. This difference resulted from the tendency of the females in the mid dose group to gain more weight than the controls during several weeks after Week 6.

Statistically significant decreases (as compared to controls) in absolute body weight were observed in females at the high dose (4 to 7% decrease)
during weeks 1-13 and also at the mid dose (2 to 4% decrease) during weeks 2-6, 8, 9, and 13. Body weight gains at the high dose were 30% below that of the control in the first week of the study and 11 to 21% below the weight gain of controls during study weeks 2-13. Weight gain for the mid dose group was 9 to 14% below those of the control group during study weeks 1-6 and 5 to 7 % below those of control during study weeks 8-10. Body weight and body weight gain at the low dose was not significantly different from controls throughout the study.

Consistent with the increase in food consumption observed during the recovery phase, the mean body weight and weight gains for the females in the
recovery group were not different from the control group at any measurement period (weeks 14 through 17).

Ophthalmic Examinations
No animals with recorded lesions in the prestudy ophthalmic examinations other than corneal dystrophy of minimal to mild intensity were used in the
study. These corneal lesions were diagnosed in the prestudy ophthalmic examination in 97% percent of the animals used in this study. The records for the prestudy examinations have been retained in the raw data. Corneal dystrophy was diagnosed in all but one of the animals (80 of 80 males and 79 of 80 females). The lesion was considered mild in most instances and occurred with approximate equal frequency across all groups. No other lesions were observed and none of the corneal lesions were related to treatment with DETA.

Clinical Pathology
Several hematology, clinical chemistry, and urinalysis measurements were altered compared to controls in animals treated with DETA. A summary of
these changes follows:
Male - Hematology
1. 15000 ppm - statistically significant increases in MCV and MCH at 6 weeks (+2%) and at 13 weeks (+3%). MCV and MCH were also significantly
elevated (+2%) in treated males in the recovery group. A statistically significant increase in reticulocyte count at 6 and 13 weeks (3.8 and 2.5% of
the RBCs respectively for the two test periods versus 1.9% of the RBCs for controls) although these counts were within the normal range for this strain
of rats (up to 4%).
2. 7500 ppm - statistically significant increases in MCV at 6 weeks (+1%) and significantly higher MCV and MCH at 13 weeks (+I% and +2%, respectively). A statistically significant increase in reticulocyte count (3.5% of the RBCs) was also observed at 6 weeks.
3. 1000 ppm - A statistically significant increase in reticulocyte count (2.3% of the RBCs) at 13 weeks.

Female - Hematology
1. Statistically significant increases in MCV in the high and mid dose groups at 6 weeks (each +1%).
2. Statistically significant increases in total leukocytes at the high and mid dose at 6 weeks (each +30%) and at 13 weeks (+31% and +22%, respectively) with associated increases in lymphocytes at the high and mid dose at 6 weeks (+33% and +32%, respectively) and at 13 weeks (+32% and +17%, respectively). In addition, increases that were not dose related were observed in segmented neutrophils in the high, mid, and low dose groups at 13 weeks (+32%, +47%, and +30%, respectively).
3. Statistically significant increases in reticulocytes in the high dose group at 13 weeks (2.7% of the RBCs versus 2.1% for the controls) and in
platelets in the high and mid dose groups at this time period.
4. Statistically significant increases from controls in MCV (+1%), erythrocytes (+2%), hemoglobin (+3%), and hematocrit (+4%), and lymphocytes (+17%) were observed in the treated females following the recovery period. Total leukocytes were also increased (+12%) although the value was not statistically significant.

A tendency toward increased blood cell counts (including total leukocytes, lymphocytes, segmented neutrophils, reticulocytes, and platelets) was observed in the treated animals (particularly females) in this study. These increased counts were, however, of a minor magnitude and the majority of these values fell within normal ranges for this strain of rat. The biological significance of these differences was, therefore, considered negligible and not related to a direct effect of DETA on the hematopoetic system. This conclusion was substantiated by the lack of histopathologic changes in the bone marrow. Changes in erythrocytes, hemoglobin, and hematocrit in females following the 4-week recovery period were also considered to be of no biological significance since there were no alterations in these measurements during the treatment period.

Female - Serum Clinical Chemistry
1. Statistically significant decreases in glucose were observed in the high and mid dose groups at 13 weeks (each -10%) compared to controls.
Glucose was also depressed in treated females following the recovery period.
2. A statistically significant decrease in albumin was observed in the high dose group at 13 weeks (-8%). No change in albumin in the treated group
after the recovery period was recorded.

Female - Urinalysis
An increase in urine pH (approximately 1 pH unit) was observed in females at the high and mid dose groups at 6 and 13 weeks. No other effects on urine measurements were observed at either test period.

No treatment-related effects were observed in serum chemistry or urinalysis measurements in male rats treated with DETA at any dose or in
hematology measurements in males treated with 1000 pprn DETA. No effects were observed in hematology, serum chemistry, or urinalysis measurements in females treated with 1000 pprn DETA.

Organ Weiphts and Final Body Weights
Males
A statistically significant difference compared to controls in body weight prior to sacrifice for males of the high dose group (-6%) was consistent with
measurements made during the study. The statistically significant decrease in absolute spleen weight and increases in organ to body weight ratios (brain, kidney, and testes) in the animals from the high dose group were consistent with and were considered to result from the decreased body weights. The statistically significant decreases in absolute brain weight in the animals from the high and mid dose groups were small and, without confirming gross or histopathologic findings, were considered spurious. No treatment-related differences in absolute or relative organ weights in males from the 1000 ppm group were recorded.

Following the recovery period, the mean body weight of treated males remained depressed (-6%). As with the 13-week results, the statistically
significant decreases in absolute organ weights (lung, spleen, and heart) or organ to brain weight ratio (lung) and increases in organ to body weight
ratios (kidney and testes) were considered related to the decreased body weights. The minor increase in testes weight as percent of brain weight was
considered of no biological significance.

Females
Statistically significant decreases (compared to control) in body weights prior to the 13-week sacrifice for females at the 7500 pprn dose (-3%) and
15000 ppm dose (-6%) were observed. These changes were consistent with the body weights measured during the study. Absolute kidney weights were significantly higher than controls in females from the 15000 pprn (+7%) and the 7500 pprn (+6%) groups. Kidney weights expressed as percent of body weight and brain weight were also increased in the high dose group (14% and 7% increases, respectively) and mid dose group (9% and 6% increases, respectively). Other statistically significant differences, expressed as percent of body weight, occurred for liver (+6%), brain (+7%), heart (+7%), and adrenals (+IS%) at the high dose and for liver (+6%) and brain (+3%) at the mid dose. No other treatment-related effects on female organ weights were observed at 15000 and 7500 ppm group throughout the study. No differences from control attributed to treatment with DETA were observed for organ weights of females from the 1000 ppm group.

There were no differences from the control group in the mean body weight prior to sacrifice for treated females sacrificed at 17 weeks, following the
4-week recovery period. Absolute kidney weights in treated recovery animals were significantly increased (+5%) at 17 weeks compared to controls. Liver and adrenal weights in treated females were significantly increased when expressed as absolute weight (+7% and +13%, respectively), as percent of body weight (+7% and +14%, respectively), and as percent of brain weight (+6% and +13%, respectively).

Gross Necropsy Findings and Histopathology
No abnormal gross necropsy findings or histopathologic findings were attributed to DETA exposure for either sex at any dose level.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 70 - <= 80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No alterations related to treatment in other measurements including food consumption, body weights, ophthalmologic findings, hematology, serum clinical chemistry, urinalysis, or organ weights were observed in the 1000 ppm group for either sex.
Dose descriptor:
LOAEL
Effect level:
>= 530 - <= 620 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No alterations related to treatment in other measurements including food consumption, body weights, ophthalmologic findings, hematology, serum clinical chemistry, urinalysis, or organ weights were observed in the 1000 ppm group for either sex.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No additional information available.       

Applicant's summary and conclusion

Conclusions:
Dietary exposure of Fischer 344 rats to 7500 or 15000 ppm of the dihydrochloride salt of diethylenetriamine resulted in dose-related systemic toxicity. In addition, the majority of the changes, particularly in male rats, did not completely recover over a 4-week post-dosing period. The no observable effect level for this species and strain over the 90-day test period was 1000 ppm.
Executive summary:

Fischer 344 rats were exposed to DETA in the diet at concentrations of 0, 1000, 7500, or 15000 ppm for 90 days. Group sizes were 30/sex for the high dose and control groups, and 20/sex for the intermediate and low dose groups. The doses corresponded to approximate mean intake levels of 70, 530, and 1060 mg/kg/day for the males and 80, 620, and 1210 mg/kg/day for the females of the 1000, 7500, and 15000 ppm groups, respectively. Following the exposure period, ten animals from each of the control and high dose groups were observed for 4 weeks during a recovery phase. No treatment-related clinical signs, gross necropsy findings, or histopathologic findings were observed for either sex at any dose level. No alterations related to treatment in other measurements including food consumption, body weights, ophthalmologic findings, hematology, serum clinical chemistry, urinalysis, or organ weights were observed in the 1000 ppm group for either sex.

Several treatment-related effects on in-life, clinical pathology, and organ weight measurements were observed for the 7500 and 15000 ppm groups through the 13-week treatment period. Decreases in food consumption were observed intermittently throughout the dosing period in males and females at the high dose. Dose-related decreases in body weight or weight gain were observed for both sexes in the mid and high dose groups. Minimal changes in clinical pathology measurements that were potentially treatment-related included: for the males, increased MCV and MCH in the high and mid dose groups, and for the females, decreased glucose and albumin, and increased MCV in the high dose group, and similar changes in glucose and MCV in the mid dose group. In addition, increased urine pH in the females from the high and mid dose groups, possibly associated with an increase in kidney weight and/or excretion of the test material, was observed after 13-weeks of exposure. A treatment-related increase in kidney weight was observed in the female rats treated with 7500 or 15000 ppm DETA. In addition, liver (mid and high dose groups) and adrenal (high dose group only) weight increases in the females may have been a result of treatment. No other statistically significant changes in any measurements were attributed to direct toxicity of DETA at 7500 or 15000 ppm dietary concentrations.

During the recovery period, female rat food consumption was greater than the concurrent control group and body weights returned to control levels. Male body weights remained lower than the controls throughout the recovery period. The majority of other alterations remained affected after the 4-week period.

In conclusion, dietary exposure of Fischer 344 rats to 7500 or 15000 ppm of the dihydrochloride salt of diethylenetriamine resulted in dose-related systemic toxicity. In addition, the majority of the changes, particularly in male rats, did not completely recover over a 4-week post-dosing period. The no observable effect level for this species and strain over the 90-day test period was 1000 ppm.