Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 NOV 1983 to 14 FEB 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

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:

yes

Remarks:

no neurobehavioural examinations

GLP compliance:

yes

Remarks:

according to US EPA GLP Standards of May 2, 1984

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

bis(6-aminohexyl)amine; hexane-1,6-diamine

EC Number:

907-605-7

Cas Number:

68815-47-4

Molecular formula:

C6H16N2 (HMD) C12H29N3 (BHT)

IUPAC Name:

bis(6-aminohexyl)amine; hexane-1,6-diamine

Details on test material:

- Name of test material (as cited in study report): BHMT; Bis-(Hexamethylene)-triamine
- Substance type: grey paste
- Stability under storage conditions: Test solutions held for up to 10 days at room temperature were found to be stable with respect to test article content.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: 42 days
- Weight at study initiation: males: 186-209 g; females: 132-152 g
- Housing: individually housed
- Diet: Certified Rodent chow #5002 (Ralston Purina Company, ST. Louis, Missouri, USA), ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean 23.5
- Humidity (%): mean 51%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test solution was prepared fresh daily at concentrations of 2,5, and 12 mg test article per ml test solution. The appropriate amount of test article was weighed and mixed with vehicle (deionized water). Additional vehicle was added to yield the required volume of prepared test article solution. Solutions were shaken by hand. Deionized water was dispensed for the control group. Test and control solutions were dispensed daily in capped containers.

- Concentration in vehicle: constant concentrations of 2, 5, and 12 mg/ml,
- Maximum dose volume applied: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions were analyzed at weeks 1, 4, 8 and 12 and were generally within ± 10% of target concentrations.
Stability of the dosing solutions was verified after storage for 1 and 10 days at room temperature.

Duration of treatment / exposure:

13 weeks (i.e. 91 days)

Frequency of treatment:

once daily, 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 50 and 120 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of range finding study (for details see SS_Ascend_IRDC_IR-83-152_1983_Repeated dose toxicity: oral 28 days)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (mortality and clinical signs)
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study initiation and in week 13
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at week 13
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight, i.e. approx. 16 hours)
- How many animals:10 rats/sexe/group
- Parameters checked: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, platelet count, reticulocyte count, differential leukocyte count, corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes (overnight, i.e. approx. 16 hours)
- How many animals: 10 rats/sexe/group
- Parameters checked: sodium, potassium, chloride, calcium, phosphorus, alkaline, phosphatase, total bilirubin, gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), ornithine carbamoyltransferase (OCT), creatine phosphokinase (CPK), urea nitrogen, creatinine, total protein, albumin, globulin (calculated), total cholesterol and glucose

URINALYSIS: Yes
- Time schedule for collection of urine: during the 16 hours fasting period before blood sampling
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: dipstick determinations of pH, protein, glucose, occult blood, nitrite, bilirubin, glucose, ketones and urobilinogen; microscopic examination of sediments and observations on color, specific gravity, volume and appearance were also performed.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following list of tissues were taken from all surviving animals and microscopically examined after processing on the control and high dose groups. For the low and mid dose groups, the liver, kidney, lung and gross lesions were sectioned for microscopic examination.
Bone (femur), Bone marrow (femur), Bone marrow smear
Brain (3 levels: fore,mid and hind brain)
Eye (2)
Gastrointestinal tract: esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum
Gonads: ovary (2) or testis with epididymis (2)
Heart
Kidney (2)
Lung with mainstem bronchi (2)
Lymph nodes: mediastinal, mesenteric
Mammary region (females only)
Pancreas
Pituitary
Prostate and seminal vesicle (2, males only)
Salivary gland, mandibular with submandibular lymph node
Sciatic nerve
Skin
Spinal cord (cervical, midthoracic and lumbar)
Spleen
Thymic region
Thyroid - parathyroid complex
Trachea
Urinary bladder
Uterus

Statistics:

Body weight (weekly), organ weight (absolute and relative to body and brain weights at terminal sacrifice), food consumption (weekly) and clinical laboratory (week 13) data were analyzed using Bartlett's test for homogeneity of variances and a one-way analysis of variance (ANOVA). Treatment groups compared to the control group, by sex, using the appropriate t-statistics (for unequal and equal variances) as described by Steel and Torrie. Dunnett's multiple comparison tables were used to assess significant differences at p < 0.05. Total bilirubin, GGT OCT, chloride and specific gravity were analyzed using a nonparametric approach, by transforming the data to ranks prior to analysis, as described by Conover and Iman.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male and one female of the high-dose group died; one male at the mid-dose died. Resppiratory rales in several animals from high- and mid-dose group.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male and one female of the high-dose group died; one male at the mid-dose died. Resppiratory rales in several animals from high- and mid-dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

in high-dose females body weight decreased compared to control group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly decreased food consumption noted in high-dose males (minus 3.5%), when food consumption was calculated on a g/kg/day basis

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Segmented neutrophils were increased in females and males of the high-dose group compared to controls, but mean values for these groups were within the normal range of historical control values for this species in this laboratory.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

related to the test item

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

related to the test item

Details on results:

CLINICAL SIGNS AND MORTALITY
One male (week 2) and one female (week 5) in the high-dose group died and one male (week 7) at the mid-dose died during the treatment period.
With the possible exception of respiratory rales seen in a few males (4/15) and females (2/15) at the high-dose level and mid-dose females (3/15),
no clinical signs of toxicity associated with treatment were observed.

BODY WEIGHT AND WEIGHT GAIN was impaired in high-dose females during the last two-thirds of the study (statistically significant at weeks 6 and 7, but maximum difference control versus high dose females minus 5%, see als table in "any other information on results")

FOOD CONSUMPTION: slightly decreased food consumption noted in high-dose males (minus 3.5%), when food consumption was calculated on a g/kg/day basis. On a g/animal/day basis, the values were comparable between the groups throughout the study

HAEMATOLOGY
Segmented neutrophils were statistically significantly increased in females of the high-dose group compared to controls. Male values in the high-dose group were also increased when compared to control values although statistical significance was not seen. In each case, mean values for these groups were within the normal range of historical control values for this species in this laboratory.

ORGAN WEIGHTS
Statistically significant variations occurred in the mean absolute weight of adrenals in male rats at 120 mg/kg/day dosage level and in the mean relative weight (to body weight) of female liver at 50 mg/kg/day dosage level. There were no test item related microscopic changes in these organs and therefore, these variations were considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test item related changes were observed in any of the tissues from male and female rats evaluated histopathologically from the treatment groups. All changes seen were considered spontaneous or incidental in nature and unrelated to the administration of the test item. A frequently observed lesion in this study was interstitial pneumonia (this was also the cause of death of the animal in the mid-dose group). Other non-treatment related changes occurred in eye, heart, kidney, liver, pituitary, prostate, testis, trachea, urinary bladder, adrenal, brain, ovary and uterus. Interstitial pneumonia was slightly increased in treatment groups but was considered non-specific and unrelated to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 3 animals (female) with respiratory effects were noted at 50 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Body weights at study termination

	Group mean body weights in g (% difference from vehicle control)
Dosage level (mg/kg/day)	males	females
0 (control)	504	281
20	491 (minus 2.6)	286 (plus 1.8)
50	494 (minus 2.0)	276 (minus 1.9)
120	498 (minus 1.2)	267 (minus 5.0)

Applicant's summary and conclusion

Conclusions:

The test material was subjected to test subchronic toxicity in rats. For 13 weeks (7days/week) the test substance was applied orally (gavage) to rats (15 males and 15 females/dose) in doses of 0, 20, 50, and 120 mg/kg bw/day. Based on the respiratory effects noted in 3 animals at 50 mg/kg bw/day the NOAEL in this study was conservatively set to be 20 mg/kg bw.

Executive summary:

Male and female Sprague-Dawley rats were subjected to test subchronic toxicity of the submission substance. Doses of 0, 20, 50, and 120 mg/kg bw/day (15 males and 15 females /dose) were applied orally via gavage at 7days/week for 13 weeks.

One male and one female in the high-dose group died and one male at the mid-dose died during the treatment period. With the possible exception of respiratory rales seen in a few males and females at the high-dose level and mid-dose females, no clinical signs of toxicity associated with treatment were observed. The high-dose females showed a slight decrease in body weights in comparison to controls (minus 5% only). No significant weight differences were measured in males at any dose level or females at the low- and mid-dose levels. Some evidence of reduced food consumption was noted for high-dose males (on the basis of mg/kg/day). Ophthalmoscopic examination did not reveal any effects of treatment on the eye. With the exception of an increase in segmented neutrophils in high-dose males and females, there was no evidence of treatment-related changes in the hematologic, clinical chemistry or urinalysis examinations. Nevertheless the values were within the historical control range for this species in this laboratory. The incidental findings from gross necropsy did not appear to be related to treatment. Males at the high-dose level had decreased absolute adrenal weights and the liver weight relative to body weight for mid-dose females was elevated. These changes were not correlated with any microscopic findings and were considered unrelated to treatment. No other significant organ weight differences between control and treatment groups were noted. The microscopic lesions observed were generally comparable in treated and control group animals and were considered to be of spontaneous origin. Interstitial pneumonia was slightly increased in treatment groups but was considered non-specific and unrelated to treatment. Therefore, treatment did not result in histopathologic changes in any organ or tissue.

CONCLUSIONS Administration of the submission substance at dose levels of 20 mg/kg bw/day did not produce any toxicological signs which were attributable to treatment. At 50 and 120 mg/kg bw/day, respiratory rales were seen in males and females. At 120 mg/kg bw/day the females had slightly decreased body weights and both males and females showed increases in segmented neutrophils which may have been treatment-related but the values were in the normal range of historical control data. Overall the no-observable adverse effect level was considered to be 20 mg/kg bw/day for both sexes.

As the only organ showing macroscopic and microscopic findings related to the substance was the lung it is possible that the effects were consecutive to the inhalation route (false route perhaps du to the viscosity of the substance).

Based on this study the HMD heavies oral systemic toxicity appears to be low.