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Toxicological information

Repeated dose toxicity: oral

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Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 weeks
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: publication, not the actual study report

Data source

Reference Type:

Materials and methods

Principles of method if other than guideline:
After an acclimatization period of 10, 17 or 24 days, weanling Wistar rats were randomly assigned to groups each of 10 male and 10 female 10 rats. The rats were fed diets containing 0, 200, 2000 or 5000 ppm putrescine. Diets and tap-water were given ad lib. for a period of 5-6 wk.
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
Details on test material:
1,4-diaminobutane-dihydrochloride (putrescine) (tool. wt 161.08 g/tool, Ref.No. 32810, purity > 99%), from Fluka AG (Buchs, Switzerland).

Test animals

Details on test animals or test system and environmental conditions:
Wistar-derived SPF-bred rats (Cpb:WU; Wistar random or Bor:WISW) were obtained from TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands or from F.Winkelmann Institute for the Breeding of Laboratory Animals GmbH & Co. KG, Borchen, Germany. They were housed conventionally under barrier conditions, in suspended stainless-steel cages fitted with wiremesh floor and front, two rats per cage in the acute studies and five rats per cage in the subacute studies.
The room temperature was maintained at 22 + 3°C and the relative humidity at 55 + 15%. Artificial light was provided continuously, for 12 hr/day from 07.30 hr until 19.30 hr. The number of air changes was about 10/hr.

Administration / exposure

Route of administration:
oral: feed
other: diets with different concentrations putrescine
Details on oral exposure:
After an acclimatization period of 10, 17 or 24 days, weanling Wistar rats (Bor:WISW) were randomly assigned to groups each of 10 male and 10
female 10 rats. The rats were fed diets containing 0, 200, 2000 or 5000 ppm putrescine. Diets and tap-water were given ad libitum for a period of 5-6 weeks.
Details on analytical verification of doses or concentrations:
The rats were weighed once weekly, and observed daily for condition and behaviour. Food intake was measured weekly, on a cage basis by weighing the feeders. Water intake was determined daily during the first week of the study in a similar fashion. Systolic blood pressure was measured by an indirect tail-cuff method (Kuijpers et al., 1986). During the acclimatization period, the rats were accustomed to the procedures involved in measuring blood pressure. Systolic blood pressure of all rats was measured once prior to the start of the study and twice per week during the morning (males on days 0, 3, 5, 10, 12, 17, 19, 24 and 26 and fi,~males on days 0, 4, 6, 11, 13, 18, 20, 25 and 27). Four successive systolic blood pressure readings were taken of each rat on the days of measuring. The mean value of the last three successful readings from each rat was regarded as the systolic blood pressure of that rat for that day.
Duration of treatment / exposure:
5 - 6 weeks
Frequency of treatment:
diet (with putrescine concentrations) and water: daily ad libitum
Doses / concentrations
Doses / Concentrations:
0, 200, 2000 or 5000 ppm
No. of animals per sex per dose:
10 male, 10 female per dose group
Control animals:
yes, plain diet


Observations and examinations performed and frequency:
Haematology and clinical chemistry.
Blood samples were collected from the tip of the tail of all rats early in wk 5 andexamined for haemoglobin concentration, packed cell volume and erythrocyte, leucocyte and thrombocyte counts (Sysmex K-1000 Haematology Analyzer, Toa Medical Electronics Co, Ltd, Kobe, Japar~), differential leucocyte count (blood smears stained according to Pappenheim) and prothrombin time (Normotest, Nyegaard & Co. A/S, Oslo, Norway). Whole blood taken from all rats after overnight fasting in wk 5 was examined for glucose (Boehringer Glucoquant No. 245-178; Boehringer Mannheim GmbH, Mannheim, Germany). Blood samples taken from the abdominal aorta of all rats at autopsy were centrifuged at 1250 g for 15 min and then analysed for alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), 7-glutamyl transferase, total protein, albumin, urea, creatinine, total bilirubin, calcium and inorganic phosphate (Cobas-Bio Centrifugal Analyzer, Hofmann-La Roche, Basle, Switzerland), chloride (Chloro Counter, Marius, Utrecht, The Netherlands) and sodium and potassium (Electrolyte-2 Analyzer, Beckman Instruments, Brea, CA, USA).

In wk 5 all rats were deprived of water for 24 hr and of food for 16 hr. Urine was collected during the last 16 hr of the deprivation period and its
volume (calibrated tubes) and density (refractometer; Bellingham and Stanley, London, UK) were measured.

Sacrifice and pathology:
The rats were killed in wk 5 or 6 by exsanguination from the abdominal aorta while under light ether anaesthesia, and a thorough autopsy was performed. The following organs of each rat were weighed and the organ/body weight ratios were calculated: adrenal:~, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thymus, thyroid and uterus. Samples of these organs and of the coagulating glands, epididymides, seminal vesicles, spinal cord, mesenteric lymph nodes, prostate, peripheral nerve, spinal cord and gross lesions were fixed in 10% neutral buffered (pH 7.0) formalin, embedded in paraffin wax, sectioned at 5 #m, and stained with haematoxylin and eosin. Detailed microscopic examinations were carried out on all above-mentioned organs of all rats of the control and high-dose groups. The kidneys, liver, testes, spleen, thymus and heart of all rats of the low- and mid-dose
groups in the spermine study were also examined, because effects were noted in the high-dose rats. A thorough autopsy was also performed on rats that were found dead or were killed when moribund during the study. The organs of these animals were not weighed, but tissues were preserved and examined microscopically.
Statistical analysis
Data on body weight were evaluated by one-way analysis of covariance, followed by Dunnett's multiple comparison tests. The laboratory determinations and organ weights were evaluated by one-way analysis of variance, followed by Dunnett's multiple comparison tests. The differential white blood cell counts were analysed by Kruskall-Wallis non-parametric analysis of variance followed by Mann-Whitney U-tests. Data on food and water intake were evaluated by analysis of variance, followed by least significant difference tests (experimental unit: the cage). Pre and post treatment data on blood pressure were analysed using paired t-tests. The mortality incidence and the histopathological changes were examined by Fisher's exact probability
test. All comparison tests were two-tailed, and a probability level of P < 0.05 was considered significant.

Results and discussion

Results of examinations

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased body weights associated with diminished food intake and sometimes also with decreased food efficiency were observed in the top-dose group.
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
putrescine showed no evidence of renal toxicity in the present studies, not even at the high-dose levels.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Details on results:
Putrescine. Mean body weights, food intake and food efficiency were slightly decreased with 5000 ppm in both sexes, although the differences from the controls were statistically significant for body weights in females and for food intake in males.
Plasma alanine amino-transferase activity was slightly increased in females of the 5000 ppm group.
In the 5000 ppm group, the relative weight of the brain was significantly increased in females.

Effect levels

Dose descriptor:
Effect level:
ca. 180 mg/kg bw/day (actual dose received)
Based on:
test mat.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

In the oral feeding studies no decreases in systolic blood pressure were observed.

Applicant's summary and conclusion

No dose related significant effects were reported after a 6 week feeding study. Decreased body weights associated with diminished food intake were seen in the hight dose group. The NOAEL was 2000 ppm (180 mg/kg bw/day)
Executive summary:

Male and female rats were fed diets containing 0, 200, 2000 or 5000 ppm putrescine. Diets and tap-water were given ad libitum for a period of 5-6 weeks. Decreased body weights associated with diminished food intake were seen in the high dose group.

The no-observed-adverse-effect level (NOAEL) was 2000 ppm (180 mg/kg body weight/day).