N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Effects of Diethylenetriamine Dihydrochloride Following 13 Weeks of Dietary Dosing in Fischer 344 Rats

Author:

Leung HW and van Miller JP

Year:

1997

Bibliographic source:

Food Chem. Toxicol., 35, 481-487

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diethylenetriamine dihydrochloride

IUPAC Name:

Diethylenetriamine dihydrochloride

Constituent 2

Reference substance name:

3488-90-2

Cas Number:

3488-90-2

IUPAC Name:

3488-90-2

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Diethylenetriamine dihydrochloride
- Preparation: Diethylenetriamine dihydrochloride (DETA, 2HCI; CAS No. 3488-90-2) was prepared by reacting diethylenetriamine (DETA; CAS No. 111-40-0) with commercial concentrated hydrochloric acid in a solvent mixture of ethanol and toluene. Water was removed continuously from the reaction product as the known toluene-ethanol-water azeotrope. Makeup solvent was added at an equal rate to replace that being distilled from the solution until the product gradually crystallized from the solution. The purity of the product was assayed, analysing for chloride and for free and total amine. Free DETA was extracted by adding 50% aqueous NaOH, centrifuging, and drawing off the upper layer for analysis by gas chromatography-mass spectrometry.
- Molecular weight: 176.0886
- Smiles notation (if other than submission substance): [H+].[H+].C(NCCN)CN.[Cl-].[Cl-]
- Analytical purity: > 99%

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 (Kingston, NY, USA)
- Age at study initiation: 6 weeks
- Weight at study initiation: body weight ranges of the males and females at first dose were 105.2-145.5g and 92.1-108.8g, respective! y.
- Fasting period before study: none
- Housing: one per side of divided stainless-steel cages
- Diet: ground, certified Rodent chow No. 5002 (Ralston Purina, St Louis, MO, USA) ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 66-77°F
- Humidity (%): 40-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: rat chow

Details on oral exposure:

DIET PREPARATION
Diets were prepared once weekly by mixlng rat chow with DETA.2HCI in a grinder for 30 min to produce a 30,000 ppm premix. Diets of various DETA.2HCl concentrations were prepared by diluting and further blending of this premix with the appropriate amounts of ground chow.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Experimental diets were analysed using HPLC. Homogeneity and stability of the diets were established prior to the start of the study. Homogeneity studies were performed on nine samples (three each from the top, middle and bottom of the mixing bowl) from each diet concentration. The range of the analyses was 89.4 to 109.5% of nominal. Stability studies indicated that DETA.2HCI remained stable for at !east 7 days and 21 days when stored at room temperature in open glass feeder jars and in closed polyethylene containers, respectively. Concentrations were verified for ali diets each week for the first 4 wk of the study and, in subsequent weeks, concentrations concentrations were verified for one diet selected sequentially. The analytical concentrations ranged from 90.6 to 108.6% of nominal.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Ad libitum

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1000, 7500 and 15000 ppm DETA, HCl
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
70, 530 and 1060 mg DETA, HCl/kg bw/d (41, 307 and 614 mg DETA/kg bw/d) for the males
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
80, 620 and 1210 mg DETA, HCl/kg bw/day (46, 360 and 701 mg DETA/kg bw/d) for the females
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: A preliminary study with 10 rats per sex per group fed diets containing DET A.2HCI at concentrations of 0, 5000, 10,000, 25,000 or 50,000 ppm for 14 days was conducted to aid in the selection of doses for the present study. There was no mortality in any group, but significant toxicity was observed in the two highest dose groups. Signs of toxicity included decreases in food consumption, body weight and weight gain, and organ weights (liver,
kidneys, spleen, heart and lungs). At 10,000 ppm, decreases in body weight gain, and in Jung and liver weights were noted in males only. The only effect noted at 5000 ppm was decreased Jung weight in males.
- Post-exposure recovery period in satellite groups: the high dose and control groups contained an additional 10 rats per sex that were designated for a 4-wk recovery period.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, once daily for overt clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to dosing and final killing
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before dosing, and in wk 6 and 13 of dosing
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes, about 22 hr
- How many animals: 10 per sex per group + recovery animals
- Parameters: Leucocyte count, Erythrocyte count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration, Platelet count, Differential leucocyte count, Reticulocyte count, Prothrombin time, Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before dosing, and in wk 6 and 13 of dosing
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes, about 22 hr
- How many animals: 10 per sex per group + recovery animals
- Parameters : Glucose, Urea nitrogen, Creatinine, Total protein, Albumin, Globulin, Total bilirubin, Direct bilirubin, Indirect bilirubin, Cholesterol, Creatinine kinase, Aspartate aminotransferase, Alanine aminotransferase, Lactic dehydrogenase, Alkaline phosphatase, Calcium, Phosphorus, Sodium, Potassium, Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: for 24 hr from 10 rats per sex per group in wk 6 and 13.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters: Volume, Specific gravity, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen, Microscopic elements

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

After l3 and 17 (recovery groups) wk of the study, rats were weighed, anaesthetized with methoxyflurane and killed by severing the brachial vessels to permit exsanguination. A complete autopsy was performed.

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
brain, liver, kidneys, lungs, spleen, heart, adrenals, thyroid and gonads

HISTOPATHOLOGY: Yes
A histological examination covering an extensive range of tissues was performed on control and high dose (15,000 ppm) animals killed in wk 13. The lungs, liver, kidneys and gross lesions only were examined for the 1000 and 7500 ppm groups, as weil as the controls and recovery groups killed at wk 17

Statistics:

Data for continuous, parametric variables were compared with the control group by the 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 the analysis of variance of unequal variances, followed if necessary, by separate variance t-tests. Non-parametric data were analysed 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 test where appropriate. The fiducial limit of P < 0.05 (twotailed) was used as the critical leve! of significance for ali 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:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

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:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
A single female in the 1000 ppm group died in the wk 8. The cause of death was not apparent from the gross and microscopie examination.
There were no other clinical signs related to DETA.2HCl treatment in all groups over the 90-day exposure period.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain were reduced compared with controls in the 15,000 ppm males and females throughout the study. The male rats showed only minimal recovery, and their body weights and body weight gains remained lower than controls through wk 17. In contrast, body weights and weight gains of the female rats, consistent with their increase in food consumption during the recovery period, did not differ from those of the controls at the end of the recovery period. At 7500 ppm, body weights were reduced in wk 2, 3, 6 and 9-13 in males and wk 2-6, 8-9 and 13 in females. Body weight gains were reduced throughout the study in males, and in the wk 1-6, 8-10 and 13 in females. There were no treatment-related changes in body weight or body weight gain at 1000 ppm in either sex.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Statistica!ly significant reductions in food consumption compared with controls were observed in the 15,000 ppm males in wk 1-4, 6-7, 9-10 and 12-13, and in the 15,000 ppm females in wk 1-5. A statistically significant decrease was observed in the 7500 ppm males in wk 1 and in the 1000 ppm females in wk 5-6. During the recovery phase (wk 14-17), food consumption was increased relative to control in the females but not the males.
On the basis of the food consumption and body weight data, the dietary concentrations of 1000, 7500 and 15,000 ppm corresponded to mean doses of 70, 530 and 1060 mg/kg/day for the males and 80, 620 and 1210 mg/kg/day for the females.

OPHTHALMOSCOPIC EXAMINATION
Prestudy ophthalmic examinations indicated that all but seven of the rats had minimal to mild corneal dystrophy. Because of the historically high incidence of this lesion for this strain of rats from all animal suppliers, and the lack of potential impact on interpreting the toxicity of DETA.2HCI, these animals were accepted for study despite the presence of this corneal lesion. In the terminal ophthalmic examination, corneal dystrophy was diagnosed in all but one animal. The lesions were again mild and occurred with equal frequency across all groups. No other ocular lesions were observed.

HAEMATOLOGY
At wk 6, increases in mean corpuscular volume (MCV) in the 7500 and 15,000 ppm males and increases in mean corpuscular haemoglobin (MCH) in the 15,000 ppm males were observed. The MCV and MCH in the 7500 and 15,000 ppm males at wk 13 and in the 15,000 ppm males at wk 17 (recovery) were elevated. In all cases, the magnitude of these changes was 3% or less compared with control. These changes were related to statistically significant increases in reticulocytes, observed at wk 6 and 13. Increases in MCV were also observed in the 7500 and 15,000 ppm females at wk 6, and in the 15,000 ppm females at wk 17.
A statisticaliy significant increase in total leucocyte count, attributed to a statisticaliy significant increase in lymphocytes, was observed in the 7500 ppm and 15,000 ppm females at wk 6 and 13. At wk 13, statisticaliy significant increases in segmented neutrophils also contributed to the increase
in total leucocyte numbers. Although total leucocytes were no longer significantly altered following a 4-wk recovery, the lymphocyte increase was still
statisticaliy significant.

CLINICAL CHEMISTRY
The serum glucose concentrations were depressed in females exposed to 7500 and 15,000 ppm at wk 13, and persisted foliowing a 4-wk recovery period.

URINALYSIS
Urine pH was significantly increased in the 7500 and 15,000 ppm groups at wk 6 and 13. This alteration, however, was no longer observed foliowing the recovery period.

ORGAN WEIGHTS
Statisticaliy significant increases in absolute kidney and brain weights and in relative kidney, brain and testes weights were observed in the males at
15,000 ppm. The relative kidney and testes weights remained elevated foliowing the 4-wk recovery period. The females showed a significant, dose-related increase in relative kidney, brain, and Iiver weights at 7500 and 15,000 ppm, and in relative heart and adrenal weights at 15,000 ppm. Following
the recovery period, only the relative Jiver and adrenal weights remained elevated.

GROSS PATHOLOGY
One female rat from the 1000 ppm group died on study day 55. This rat was found to be emaciated at autopsy, but the specifie cause of death could not be determined. There were no treatment-related gross findings in any group.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no statistically significant differences in the incidences of microscopic findings between the 15,000 ppm and control groups killed at wk 13 or 17.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: depressed body weights and minor alterations in clinical pathology measurements and organ weights. NOAEL corresponding to 41 mg DETA/kg bw/d in male rats and 46 mg DETA/kg bw/d in female rats

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In summary, dietary exposure to DETA.2HCI at 7500 and 15,000 ppm for 90 consecutive days resulted in systemic toxicity, as indicated by depressed
body weights and minor alterations in clinical pathology measurements and organ weights. A no-observed-effect leve! of 1000 ppm (corresponding
to 41 mg/kg/day in male rats and 46 mg DETA/kg/day in female rats) was established in this study.

Executive summary:

Fischer 344 rats were fed a diet containing the dihydrochloride salt of diethylenetriamine (DETA, 2HCl) at concentrations of 1000, 7500 or 15,000 ppm for 90 consecutive days. Based on food consumption and body weight, the mean corresponding dosages were 70, 530 and 1060 mg/kg/day for the males and 80, 620 and 1210 mg/kg/day for the females. Decreases in food consumption were observed intermittently throughout the dosing period in both sexes at 15,000 ppm. Dose-related decreases in body weight or weight gain were observed for both sexes at 7500 and 15,000 ppm. Changes in clinical pathology measurements observed at 7500 and 15,000 ppm included increases in mean corpuscular volume and mean corpuscular haemoglobin in males, and increases in mean corpuscular volume, total leucocytes and urinary pH, and a decrease in serum glucose concentration in females. The relative kidney, brain and testes weights were increased in the 15,000 ppm males. In the females, the relative kidney, brain and liver weights were increased at 7500 and 15,000 ppm, and the relative heart and adrenal weights were elevated at 15,000 ppm. There were no treatment-related clinicat signs, gross autopsy or histopathological findings for either sex at any dose level. Animals improved only slightly from the effects of treatment following a 4-wk recovery period. A no-observed-effect leve! of 1000 ppm (corresponding

to 41 mg DETA/kg/day in male rats and 46 mg DETA/kg/day in female rats) was established in this study.