Registration Dossier

Administrative data

Description of key information

A modern screening study (OECD 422) and an Repeated Dose Toxicity Study (OECD 408) are available to address repeated dose toxicity.

A modern screening study (OECD 422) and an Repeated Dose Toxicity Study (OECD 408) are available to address repeated dose toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern, guideline and GLP-compliant study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 9 - 10 weeks old.
- Weight at study initiation: 195 - 212g for males and 156 - 164g for females.
- Fasting period before study: Groups were food deprived prior to the start of testing.
- Housing: From arrival to pairing animals were housed up to 5/sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor.
During mating animals were housed one male to one female in clear polycarbonate cages measuring 42.5x26.6x18 cm with a stainless steel mesh lid and floor.
After mating the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages measuring 42.5x26.6x18 cm for the gestation period, birth and lactation.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet was offered ad libitum throughout the study, except to those groups which were food-deprived during testing.
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 25 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light (artificial light).

IN-LIFE DATES: From 05 March 2012: To: 28 April 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of N,N,N´,N´-Tetramethyl-1,6-hexanediamine was dissolved in the vehicle (purified water) and brought to the final volume appropriate for each concentration (concentrations of 0.75, 2.25 and 7.5 mg/mL) and shaken manually until complete dissolution. The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable. Results of the analyses were within the limits of acceptance. The stability was found to be 24 hours at room temperature in the concentration range of 0.75 to 7.5 mg/mL.
Samples of the formulations prepared on Week 1 and last Week (when all females were present) were also analysed to check the concentration. Results of the analyses were within the limits of acceptance.
Chemical analysis was carried out in the range from 1.0 to 2.5 mg/mL. In the present study the validation of the method was performed for the lower concentration 0.1 mg/mL.
Duration of treatment / exposure:
Male test animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and up to the day before necropsy.
Female test animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum.
Frequency of treatment:
Daily during the exposure period
Remarks:
Doses / Concentrations:
0, 7.5, 22.5, 75.0 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Positive control:
Not applicable; not required for this type of study
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination.
Each animal was removed from the home cage and observed in an open arena for a minimum of 3 minutes. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the necropsy procedure.
- Anaesthetic used for blood collection: Yes- isofluorane anaesthesia
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked include: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differetntial leucocyte count (including neutrophils, lymphocytes, eosinophils, basophils, monocytes and large unstained cells) and platelets.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During the necropsy procedure.
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked include: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, phosphorous, total biliubin, total cholesterol, bile acids, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium and chloride.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the study, towards the end of treatment.
- Dose groups that were examined: males and 5 females were randomly selected from each group for examination.
- Battery of functions tested: sensory activity, grip strength and motor activity.

OTHER:
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made to determine any anomalies of the oestruss cycle and the pre-coital interval.

Parturition check and duration of gestation: A parturition check was performed from Day 20 to Day 25 post coitum. Gestation periods were taken as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

Hormone determinations: Approximately 1.2 mL of blood samples were drawn under isofluorane anaesthesia from the same animals in the food-deprived group. Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature.
The serum obtained was divided in several aliquots and frozen at -80°C.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All females were examined for external and internal abnormalities, the number of visible implantation sites (pregnant animals) and the number of corpura lutea (pregnant animals). The uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Samples of all the tissues (all parental animals) were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).

HISTOPATHOLOGY: Yes. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.

The histopathological examination was restricted to the cervix, clitoral gland, ovaries, uterus and vagina from all parental females, the coagulating gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes from all parental males, tissues selected from 5 males and 5 females selected at random in the control and high dose groups killed at term and all abnomalities in all groups.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if ‘n’ was more than 5.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs including hunched posture, piloerection and pale aspect were observed in one female at 75 mg/kg bw/d
Mortality:
mortality observed, treatment-related
Description (incidence):
Clinical signs including hunched posture, piloerection and pale aspect were observed in one female at 75 mg/kg bw/d
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
One female at 75 mg/kg bw/d showed lower body weight gain towards the end of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the highest dose level of 75 mg/kg bw/d, decreased eosinophil counts were observed in both sexes. Anaemia, leucocytosis and thrombocytopenia secondary to gastric irritation were seen in one female at the highest dose level.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Ulceration and chronic inflammation at the stomach were seen in one female at the highest dose level. In addition, a moderate atrophy of the thymus was also observed. The latter change could be considered to be stress related.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred throughout the study. At the daily clinical examination treated males did not show any relevant clinical signs throughout the study.
Prior to the mating period one female receiving the highest dose level (75 mg/kg/day) showed rales for 2 days.
No relevant clinical signs were noted in control and treated females during the post coitum period.
Red staining on the cage tray was observed on Day 23 post coitum in female one female receiving 7.5 mg/kg/day.
One female receiving 75 mg/kg/day showed hunched posture, piloerection and pale aspect during the post partum period.

BODY WEIGHT AND WEIGHT GAIN

Means of body weight and body weight gain were comparable between control and treated groups both in males and females throughout the study. Reduced weight gain was seen in one high dose female.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

Food consumption was unaffected by treatment in both sexes during the study.

HAEMATOLOGY

A marked decrease of eosinophils was seen in animals of both sexes receiving 75 mg/kg/day. In 4/5 of the females no eosinophils were detected.


CLINICAL CHEMISTRY

There were no effects of treatment.

NEUROBEHAVIOUR
Motor activity recorded at the end of treatment was comparable between control and treated groups. Variations recorded in the sensory reaction to stimuli measurements, performed at the end of the treatment period, were considered of no toxicological significance.

ORGAN WEIGHTS
A decrease in absolute brain weight in males receiving 7.5 mg/kg/day was observed. In females receiving 22.5 mg/kg/day or more, there was a dose-related decrease in absolute thyroid weight. There was a decrease in relative spleen weight in males receiving 75 mg/kg/day. Females treated with 22.5 mg/kg/day showed a decrease in relative thyroid weight.

GROSS PATHOLOGY

Reduced size of prostate glands was observed in three males of each treated group associated with reduction of seminal vesicles and testis (left) in one low dose male. Since there was no dose-dependency and in the absence of histopathological correlates, these findings were considered not to be treatment-related. One female at 75 mg/kg bw/d showed thickened glandular and non glandular stomach, dark red areas and dark contents at the stomach.

HISTOPATHOLOGY

Mucosal ulceration, oedema, chronic inflammation in the stomach and moderate atrophy in the thymus were observed in one high dose level female; the latter change could be considered to be stress related. No further changes were seen in selected organs/tissues evaluated in males or females
receiving N,N,N´,N´-Tetramethyl-1,6-hexanediamine which could be related to treatment. The lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.

A detailed qualitative examination of the testes was performed on 5 randomly selected animals in control and high dose groups respectively. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
The PAS-stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

OTHER FINDINGS
Oestrus Cycle, reproductive parameters, pairing combination and mating performance: Oestrus cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups.

Implantation, pre-birth loss data and gestation length of females: Gestation periods were similar in treated groups and controls. All dams with the exception of
one of the low dose group in which no pups were found gave birth between Days 22 and 23 post coitum. Corpora lutea, implantations, total litter size and pre-birth loss (percentage) were similar in control and treated groups.

Litter data at birth on Day 1 and on Day 4 post partum of females and sex ratio of pups:No differences in total and live litter size, mean pup weight and sex ratio were noted between groups at birth and on Day 4 post partum.
Mean litter weight was lower than control group both on Day 1 post partum and on Day 4 post partum in females receiving 75 mg/kg/day. The change reached statistical significance on Day 4 post partum. In addition, sex ratio was comparable between control and treated groups.

Clinical signs of pups: Cold to touch, apparently no food intake and smaller than others were in general the clinical signs noted in control and treated pups.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum: No milk in stomach was noted at necropsy in the decedent pups.
Pups sacrificed on Day 4 post partum showed no milk in stomach with similar incidence both in control and treated groups.
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
22.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed at the highest dose level of 75 mg/kg bw/d
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed at the highest dose level of 75 mg/kg bw/d
Critical effects observed:
not specified

Haematology Parameters - Group mean data – Males:

Parameters (units)

 

Group

1

2

3

4

Red blood cell count (106/ul)

Mean

SD

n

8.416

0.462

5

8.302

0.326

5

8.522

0.330

5

9.028

0.871

5

HAEMOGLOBIN

(g/dl)

Mean

SD

n

14.86

0.45

5

14.62

0.56

5

15.16

0.39

5

15.24

1.58

5

Haematocrit (%)

Mean

SD

n

44.22

0.91

5

43.72

1.70

5

45.80

1.67

5

48.04

7.07

5

Mean Red Blood Cell Volume (fl)

Mean

SD

n

52.64

2.38

5

52.66

1.62

5

53.74

0.79

5

53.04

3.55

5

Mean Corpuscular HB

(pg)

Mean

SD

n

17.70

1.23

5

17.62

0.86

5

17.80

0.42

5

16.90

0.99

5

Mean Corpuscular HB

(g/dl)

Mean

SD

n

33.58

0.90

5

33.46

0.88

5

33.12

0.63

5

31.88

1.44

5

Reticulocytes %

Mean

SD

n

1.668

0.192

5

2.096

0.243

5

1.954

0.214

5

2.186

0.418

5

 

Reticulocytes (109/L)

Mean

SD

n

139.90

12.59

5

173.78

18.48

5

166.80

20.15

5

197.86

45.97

5

White blood cell count (103/ul)

Mean

SD

n

5.854

1.044

5

4.354

0.791

5

4.912

1.188

5

6.568

1.110

5

Neutrophils (103/ul)

Mean

SD

n

0.852

0.202

5

1.070

0.304

5

1.058

0.332

5

1.174

0.323

5

Lymphocytes (103/ul)

Mean

SD

n

4.700

0.956

5

3.004

0.598

5

3.606

1.083

5

5.120

1.039

5

Monocytes (103/ul)

Mean

SD

n

0.154

0.033

5

0.160

0.041

5

0.110

0.049

5

0.154

0.069

5

Eosinophils (103/ul)

Mean

SD

n

0.064

0.015

5

0.060

0.035

5

0.048

0.015

5

0.018

0.008

5

Basophils (103/ul)

Mean

SD

n

0.034

0.017

5

0.024

0.009

5

0.028

0.015

5

0.050

0.026

5

 

Large unstained cells (103/ul)

Mean

SD

n

0.050

0.042

5

0.040

0.021

5

0.066

0.038

5

0.054

0.032

5

Neutrophils (%)

 

Mean

SD

n

14.76

3.38

5

24.70

5.94

5

22.46

7.51

5

18.22

5.64

5

Lymphocytes (%)

Mean

SD

n

80.00

4.06

5

68.92

5.50

5

72.50

8.02

5

77.66

4.85

5

Monoocytes (%)

Mean

SD

n

2.38

0.41

5

3.68

0.58

5

2.22

0.81

5

2.24

0.63

5

Eosinophils (%)

Mean

SD

n

1.16

0.44

5

1.28

0.58

5

0.96

0.18

5

0.30

0.12

5

Basophils (%)

Mean

SD

n

0.58

0.18

5

0.54

0.11

5

0.60

0.21

5

0.74

0.21

5

Large unstained cells (%)

Mean

SD

n

0.82

0.60

5

0.88

0.46

5

1.30

0.58

5

0.84

0.43

5

Platelets (103/ul)

Mean

SD

n

788.0

79.4

5

729.6

59.7

5

733.6

46.2

5

827.4

91.7

5

 

Haematology Parameters - Group mean data – Females:

Parameters (units)

 

Group

1

2

3

4

Red blood cell count (106/ul)

Mean

SD

n

6.734

0.336

5

6.750

0.186

5

7.24

0.199

4

6.000

2.445

5

HAEMOGLOBIN

(g/dl)

Mean

SD

n

12.98

0.40

5

12.78

0.64

5

13.65

0.31

4

11.54

4.77

5

Haematocrit (%)

Mean

SD

n

38.62

1.43

5

37.98

1.19

5

40.40

1.02

4

34.52

13.44

5

Mean Red Blood Cell Volume (fl)

Mean

SD

n

57.40

1.91

5

56.26

1.16

5

55.78

1.09

4

58.52

3.24

5

Mean Corpuscular HB

(pg)

Mean

SD

n

19.32

0.46

5

18.96

0.67

5

18.88

0.38

4

19.12

0.74

5

Mean Corpuscular HB

(g/dl)

Mean

SD

n

33.68

0.70

5

33.68

0.64

5

33.85

0.17

4

32.78

2.38

5

Reticulocytes %

Mean

SD

n

5.884

1.549

5

6.826

1.206

5

4.700

0.827

4

8.900

8.297

5

Reticulocytes (109/L)

Mean

SD

n

392.34

84.41

5

459.42

73.50

5

340.18

60.82

4

372.50

48.17

5

White blood cell count (103/ul)

Mean

SD

n

3.214

1.173

5

3.166

0.665

5

3.005

0.568

4

1.680

1.625

5

Neutrophils (103/ul)

Mean

SD

n

1.008

0.377

5

1.102

0.399

5

0.830

0.198

4

1.630

1.625

5

Lymphocytes (103/ul)

Mean

SD

n

2.110

1.193

5

1.960

0.286

5

2.078

0.425

4

2.830

1.643

5

Monocytes (103/ul)

Mean

SD

n

0.048

0.020

5

0.050

0.021

5

0.050

0.008

4

0.090

0.107

5

Eosinophils (103/ul)

Mean

SD

n

0.020

0.012

5

0.022

0.011

5

0.018

0.010

4

0.002

0.004

5

Basophils (103/ul)

Mean

SD

n

0.010

0.010

5

0.008

0.004

5

0.015

0.006

4

0.014

0.005

5

Large unstained cells (103/ul)

Mean

SD

n

0.022

0.019

5

0.024

0.015

5

0.020

0.008

4

0.020

0.014

5

Neutrophils (%)

 

Mean

SD

n

37.28

22.63

5

34.08

5.44

5

27.73

4.19

4

32.04

6.56

5

Lymphocytes (%)

Mean

SD

n

59.66

22.48

5

62.74

5.84

5

68.95

4.28

4

65.40

6.80

5

 

Monoocytes (%)

Mean

SD

n

1.52

0.51

5

1.54

0.62

5

1.65

0.31

4

1.58

0.59

5

Eosinophils (%)

Mean

SD

n

0.62

0.26

5

0.64

0.15

5

0.55

0.21

4

0.06

0.09

5

Basophils (%)

Mean

SD

n

0.30

0.21

5

0.24

0.05

5

0.53

0.33

4

0.40

0.19

5

Large unstained cells (%)

Mean

SD

n

0.60

0.34

5

0.72

0.36

5

0.58

0.21

4

0.48

0.25

5

Platelets (103/ul)

Mean

SD

n

1415.2

339.4

5

1345.4

168.3

5

1088.5

138.6

4

1311.4

380.7

5

Conclusions:
Under the conditions of this study and based on the results obtained, the NOAEL for N,N,N´,N´-Tetramethyl- 1,6-hexanediamine was considered to be 22.5 mg/kg bw/d.
Executive summary:

The purpose of this study was to generate information concerning toxic effects on rats of both sexes after repeated dosing with N,N,N´,N´-Tetramethyl-1,6-hexanediamine, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days. During the in-life phase, body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption and mating performance were evaluated. Clinical pathology investigations (haematology and clinical chemistry) were also evaluated. At necropsy a detailed external and internal examination was performed. The histopathological examination, including identification of the stages of the spermatogenic cycle (males) was carried out in five males of control and high dose groups, selected randomly. In addition, reproductive organs such as coagulating gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes were examined histopathologically on all parental males. No relevant signs of toxicological significance were observed at the clinical examination including body weights and food consumption. Fertility index and copulatory index were unaffected by treatment. At haematological evaluation the only alteration noted in males receiving 75 mg/kg bw/d was a decrease in eosinophils. No adverse effects were observed at clinical chemistry examination. Reduced size of prostate glands was observed in three males of each treated group, associated with reduction of seminal vesicles and testis (left) in one low dose male. Since there was no dose-dependency and in the absence of histological correlates, these findings were considered not to be treatment-related.

Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum. During the in-life phase, body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption and mating performance were evaluated. Clinical pathology investigations (haematology and clinical chemistry) were also evaluated. The histopathological examination was carried out in five females of control and high dose groups, selected randomly. In addition, reproductive organs such as cervix, clitoral gland, ovaries, uterus and vagina were examined histopathologically on all parental females. Clinical signs of pups as well as necropsy examination of pups sacrificed at termination or unscheduled deaths were recorded. At necropsy a detailed external and internal examination was performed. Litter data, sex ratios and gestation length were recorded. One female at 75 mg/kg bw/d showed clinical signs and decreased body weight gain towards the end of the study, and did not raise its offspring properly. Haematology parameters revealed anaemia, leucocytosis, and thrombocytopenia. These effects correlate with the histopathological findings of ulceration and chronic inflammation at the stomach. In addition, a moderate atrophy of the thymus was also observed. The latter change could be considered to be stress related. It is likely that the above mentioned findings are the primary cause of the worsening of general condition of this animal towards study end. Although only one female was affected, it cannot fully be excluded that the high pH value of the test compound had contributed to the inflammation process in the stomach, either directly or secondary to a pre-existing lesion. At least, the postnatal effects on the pups were considered to be the consequence of the impaired maternal health status. Consistent with males, haematological examinations revealed decreased eosinophil values at 75 mg/kg bw/d, which may be the consequence of stress at this dose level. No further compound-related effects were noted, including mating, gestation, birth, nursing behaviour, and postnatal development of the offspring.

Based on the results of the study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 22.5 mg/kg bw/d for males and females. The NOAEL for reproductive and developmental toxicity was considered to be 75 mg/kg bw/d for males and females.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
N,N,N´,N´-Tetramethyl-1,6-hexanediamine was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water was filled up to the desired volume, subsequently mixed by shaking. The test-substance preparations were produced daily up to receipt of the results of stability analyses, afterwards preparations were produced weekly. The administration volume was 10 mL/kg body weight.
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, as a part of this study.
The stability of N,N,N´,N´-Tetramethyl-1,6-hexanediamine in deionized water at room temperature for a period of 7 days was proven. (BASF project No. 01Y0487/11Y160; see PART III, Supplement).
Concentration control was performed in all concentrations at the beginning and towards the end of the administration period (see PART III, Supplement). The samples were analyzed, the reserve samples were keep frozen (at – 20°C). The retained samples were analyzed only, if any imprecision while analysis of samples from start and towards the end of the study occurs. All samples were stored until finalization of the report. A homogeneity control analysis was not performed, because N,N,N´,N´-Tetramethyl-1,6-hexanediamine was administered as a solution in deionized water.

The values of N,N,N´,N´-Tetramethyl-1,6-hexanediamine in deionized water were found to be in the range of 90-110% of the nominal concentration at start of the administration period. Towards the end of the administration period the concentration of the mid dose preparation was higher than 110% of the nominal concentration (149.6%). This deviation to a higher concentration has no influence to the validity of the study.
These results demonstrated that the animals were not administrated with lower concentrations of N,N,N´,N´-Tetramethyl-1,6-hexanediamine in deionized water than intended (see PART III, Supplement).

Duration of treatment / exposure:
3 months
No. of animals per sex per dose:
10
Control animals:
yes
Observations and examinations performed and frequency:
3.8. CLINICAL EXAMINATIONS
3.8.1. Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.


3.8.2. Clinical signs
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.


3.8.3. Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm height). The following parameters were examined:

1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/ arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/ consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size


3.8.4. Food consumption
Food consumption was determined weekly (as representative value over 7 days) for each cage. The average food consumption/cage was used to estimate the mean food consumption in grams per animal and day.


3.8.5. Drinking water consumption
Drinking water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume.


3.8.6. Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on study day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on study day 0 was calculated as body weight change.


3.8.7. Functional observational battery
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting in the morning. At least one hour before the start of the FOB the animals were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random. A detailed description of the methods, the ranking and documentation system can be found in PART III (Supplement).



Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:

1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings


Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:

1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait
15. Activity/ arousal level
16. Feces excreted within 2 minutes (consistency/ color)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes
19. Other findings


Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:

1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test



3.8.8. Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.


3.8.9. Ophthalmoscopy
The eyes of all animals were examined prior to the start of the administration period. At the end of the administration period, i.e. study day 91, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after application of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

3.8.10. Estrous cycle determination
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.
Sacrifice and pathology:
3.10. PATHOLOGY
3.10.1. Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animal No. 32, which died intercurrently was necropsied and assessed by gross pathology as soon as possible after its death.

3.10.2. Organ weights
The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicles incl. coagulating glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands
17. Uterus with cervix


3.10.3. Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:

1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymis, left
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testis, left
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

The left testis and left epididymis of all animals sacrificed at scheduled dates was fixed in modified Davidson’s solution, whereas the right testis and epididymis was used to determine sperm parameters. The eyes with optic nerve, the testes and epididymides of the animal, which died (No. 32) were fixed in 4% neutral-buffered formaldehyde solution.

Other examinations:
3.9. CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence

The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.

The results of clinical pathology examinations were expressed in Inter¬national System (SI) units.

The following examinations were carried out in all animals per test group and sex at the end of the administration period.
3.9.1. Hematology
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Parameters and methods:

Parameter Unit Method References
Leukocyte count
(WBC) giga/L cytochemistry coupled with flow cytometry
Erythrocyte count
(RBC) tera/L flow cytometric laserlight scattering
Hemoglobin
(HGB) mmol/L cyanmethemoglobin method; according to ICSH
Hematocrit
(HCT) L/L calculation:
MCV x erythrocytes
Mean corpuscular volume
(MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram)
Operator’s Guide for Advia 120 System
Mean corpuscular hemoglobin
(MCH) fmol calculation:

hemoglobin
erythrocytes
Mean corpuscular hemoglobin concentration
(MCHC) mmol/L calculation:

hemoglobin
hematocrit
Platelet count
(PLT) giga/L flow cytometric laserlight scattering
Differential blood count % and giga/L cytochemistry coupled with flow cytometry
Reticulocytes (RET) % cytochemistry coupled with flow cytometry

Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).


Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).

Parameter and method:

Parameter Unit Method References
Prothrombin time (Hepato Quick’s test)
(HQT) seconds citrated blood with calcium thromboplastin Fischer, M. and Falkensammer, Ch.,
Klin. Wschr. 86, 577-583 (1974)


3.9.2. Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters

Parameters and methods:

Enzyme (systematic name and system number) Unit Method, wave-length and measuring temperature References
Alanine aminotransferase
(ALT)
(L-alanine: 2-oxoglutarate aminotransferase;
EC 2.6.1.2.) µkat/L kinetic UV test, 340 nm; 37°C Recommendations of the German Society for Clinical Chemistry: "Standardization of methods for determining enzyme activities in biological liquids".
Aspartate aminotransferase
(AST)
(L-aspartate: 2-oxoglutarate aminotransferase;
EC 2.6.1.1.) µkat/L kinetic UV test, 340 nm; 37°C J. Clin. Chem. Clin. Biochem. 8, 658-660 (1970);
J. Clin. Chem. Clin. Biochem. 9, 464-465 (1971);
J. Clin. Chem. Clin. Biochem. 10, 182-192 (1972)
Alkaline phosphatase
(ALP)
(orthophosphoric acid monoester phosphohydrolase;
EC 3.1.3.1.) µkat/L kinetic color test, 415 nm, 37°C Roche working instructions
-Glutamyltransferase
(GGT)
( -glutamyl) peptide: aminoacid--glutamyl-transferase;
EC 2.3.2.2.) nkat/L kinetic color test, 415 nm, 37°C Szasz, G. et al.,
J. Clin. Chem. Clin. Biochem. 12, 228 (1974)
Roche working instructions


Blood Chemistry Parameter Unit Method References
Sodium
(NA) mmol/L
Potassium
(K) mmol/L ion selective electrodes (ISE) Roche - working instructions
Chloride
(CL) mmol/L
Inorganic phosphate
(INP) mmol/L molybdate reaction Henry, R.J. in: "Clinical Chemistry", Harper and Row Publishers, New York (1974); Roche working instructions
Calcium
(CA) mmol/L o-cresolphthalein complex without deproteinization Ray Sarkar, B.C. and Chauhan, U.P.S., Anal. Biochem. 20, 155 (1967); Roche working instructions
Urea
(UREA) mmol/L enzymatic determination with the urease/ glutamate dehydro-genase method Neumann, U. and Ziegenhorn, J.: XVI, Nordiska kongressen for klinisk kemi och klinisk fysiologi 1977, Oulu, Finland; Roche working instructions
Creatinine
(CREA) µmol/L enzymatic determination with the creatininase/ creatinase /sarcosinoxidase method Guder et al., J.Clin.Chem.Clin.Biochem. 24, 889-902 (1986); Roche working instructions
Glucose
(GLUC) mmol/L hexokinase/glucose-6-phosphate dehydrogenase method Schmidt, F.H., Klin. Wschr. 39, 1244-1247 (1961); Roche working instructions
Total bilirubin
(TBIL) µmol/L DPD method Wahlefeld, A.W. et al., Scand. J. Clin. Lab. Invest. 29, Suppl. 126 (1972) Abstract 11.12; Roche working instructions
Total protein
(TPROT) g/L biuret method Luxton, R.W.;Patel, P.Keir, G.Thompson, E.J., Clin Chem 1989, 35 (8), 1731-1734
Albumin
(ALB) g/L bromocresol green method Doumas et al., Clin. Chim. Acta 31, 87 (1971); Roche working instructions
Globulins
(GLOB) g/L difference between total protein and albumin
Triglycerides
(TRIG) mmol/L enzymatic color test with lipase esterase/ glycerokinase/ glycerol-3-phosphate oxidase/4-amino-phenazone mod. method by Wahlefeld, A.W., in "Methoden der enzymatischen Analyse" [Methods of enzymatic analysis] (Bergmeyer, H.U., ed.) Vol. II, 3rd ed., Verlag Chemie Weinheim, GERMANY, pp. 1878-1882 (1974); Roche working instructions
Cholesterol
(CHOL) mmol/L enzymatic determination with cholesterol esterase/ cholesterol oxidase/4-amino-phenazone (CHOD-PAP method) Siedel, J. et al., J. Clin. Chem. Clin. Biochem. 19, 838 (1981); Roche working instructions


3.9.3. Urinalysis
The dry chemical reactions on test strips (Combur-Test 10 M; Sysmex, Norderstedt, Gerrmany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Sysmex, Norderstedt, Gerrmany).

Parameters and methods:

Parameter Method References
pH methyl red and bromothymol blue
Protein (PRO) tetrabromophenol-phthaleinethylester (TBPE)
Glucose (GLU) GOD-POD reaction Test strip book by
Ketones (KET) sodium nitroprusside Roche, Mannheim, GERMANY (1977)
Urobilinogen (UBG) p-methoxyaniline-diazonium-salt
Bilirubin (BIL) 2,5-dichloroaniline diazonium salt
Blood 2,5-dimethylhexane-2,5-dihydroperoxide, tetramethylbenzidine
Specific gravity (SP.GR.) [g/L] refractometer Hamilton or Atago operating instructions
Sediment microscopy Hallmann, L., [Clinical Chemistry and Microscopy] 10, ed., 233-246, Georg Thieme, Stuttgart, Germany (1966)
Color, turbidity (COL, TURB) by visual evaluation
Volume (VOL) [mL] graduated tubes



3.9.4. Sperm parameters
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.

Sperm motility examinations were carried out in a randomized sequence. Sperm head count (testis and cauda epididymis) were evaluated in control and high dose groups. Morphology was counted in control and high dose groups.
Statistics:
Please compare with any other information
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight salivation within two hours after treatment was observed in all male animals and 5 out of 10 female animals of test group 3 (75 mg/kg bw/d). Additionally moderate salivation within two hours after treatment was seen in 3 male out of 9 and 1 out of 10 female animals of test group 3 (75 mg/kg bw/d) and 3 male animals of test group 3 (75 mg/kg bw/d) even showed severe salivation within two hours after administration. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract.
No clinical findings were observed for male and female animals in test groups 1 and 2 (7.5 and 22.5 mg/kg bw/d).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
With one exception, no animal died or was sacrificed moribund during the study. Animal No. 32 of test group 3 (75 mg/kg bw/d) died by accident on study day 41.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was reduced during single intervals in female animals of test groups 1, 2 and 3 (7.5, 22.5 and 75 mg/kg bw/d). These alterations did not show a dose-dependency and were considered as not-treatment-related and incidental.
Food efficiency:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in rats of both sexes of test group 3 (75 mg/kg bw/d), absolute and relative eosinophil cell counts were decreased. Compared to historical control ranges, controls in the present study did have high means and the test group 3 animals did have values in the lower part of (males) or below (females) the historical control ranges (males absolute eosinophils 0.08-0.17 Giga/L; relative eosinophils 1.4-2.9 %; females: absolute eosinophils 0.06-0.10 Giga/L; relative eosinophils 1.7-2.9 %). Therefore, a treatment-related, adverse effect cannot be excluded.

In males of test group 1 (7.5 mg/kg bw/d) mean corpuscular volume (MCV) was higher compared to controls, but the alteration was not dose-dependent. Therefore, this change was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.

At the end of the administration period in males of test group 1 (7.5 mg/kg bw/d) alkaline phosphatase (ALP) activities were higher compared to controls, but the alteration was not dose-dependent. Therefore, this change was regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.

In males of test group 3 (75 mg/kg bw/d) urine volume was increased and urine specific gravity was decreased (not statistically significantly). This reflects the normal adaptation of the kidneys to increased fluid intake and was regarded as treatment-related, but not adverse. In males of test groups 1 and 3 (7.5 and 75 mg/kg bw/d) urine pH values were higher compared to controls. However, the alteration was not dose-dependent and therefore, this change was regarded as incidental and not treatment-related.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Please compare with any other information of this section!
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please compare with any other information of this section!
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
22.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified

1.1.1.1.1.   Absolute organ weights

When compared with control group 0 (=100%), the following mean absolute weight was significantly increased (statistically significant changes printed in bold):

 

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(7.5)

2

(22.5)

3

(75)

1

(7.5)

2

(22.5)

3

(75)

Kidneys

 

 

 

99%

101%

110%*

* : p <= 0.05, **: p <= 0.01

 

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

1.1.1.1.2.   Relative organ weights

When compared with control group 0 (=100%), the following mean relative organ weights were significantly increased or decreased (statistically significant changes printed in bold):

 

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(7.5)

2

(22.5)

3

(75)

1

(7.5)

2

(22.5)

3

(75)

Kidneys

 

 

 

102%

98%

107%*

Thymus

90%

103%

84%*

%

%

%

* : p <= 0.05, **: p <= 0.01

 

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

 

The significant absolute (1.532 g) and relative (0.701%) weight increase of the kidneys in females, as well as the significant relative weight decrease of the thymus (0.075%) in males were within the historical control range (kidneys absolute: 1.388 -1.619 g; relative: 0.652 – 0.721%; thymus relative: 0.070 – 0.089%) and were histomorphological comparable to their respective control organs. Therefore, these changes were not considered to be treatment-related.

1.1.1.   Histopathology

(Tables IC 10 – IC 14)

 

Treatment-related findings were observed in the brain, eyes, lungs and trachea of male and females with incidences and grading according to the table below:

 

Brain

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(7.5)

2

(22.5)

3

(75)

0

(0)

1

(7.5)

2

(22.5)

3

(75)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, choroid plexus

0

0

0

8

0

0

0

10

·        Grade 1

 

 

 

3

 

 

 

1

·        Grade 2

 

 

 

4

 

 

 

6

·        Grade 3

 

 

 

1

 

 

 

3

 

In almost all male and female animals of test group 3, the epithelium of the choroid plexus in the lateral ventricles showed a macrovesicular vacuolation ranging from minimal to moderate.

 

 

Eyes with optic nerve

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(7.5)

2

(22.5)

3

(75)

0

(0)

1

(7.5)

2

(22.5)

3

(75)

No. of animals

10

10

10

10

9

9

10

10

Vacuolation, iris

0

0

0

9

0

0

0

10

·        Grade 1

 

 

 

6

 

 

 

2

·        Grade 2

 

 

 

2

 

 

 

4

·        Grade 3

 

 

 

1

 

 

 

4

 

The posterior epithelial layer of the iris showed a minimal to moderate diffuse vacuolation of macrovesicular type.

 

 

Lungs

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(7.5)

2

(22.5)

3

(75)

0

(0)

1

(7.5)

2

(22.5)

3

(75)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, bronchial epithelium

0

0

0

9

0

0

0

10

·        Grade 1

 

 

 

6

 

 

 

8

·        Grade 2

 

 

 

3

 

 

 

2

Degeneration/

regeneration

0

0

0

1

0

0

0

1

·        Grade 1

 

 

 

1

 

 

 

1

 

A multifocal macrovesicular vacuolation ranging from minimal to slight was observed predominantly in the epithelium of large to medium-sized bronchi and less frequently in terminal bronchioles. Furthermore, in one male and female of test group 3, the vacuolation was associated with minimal areas of epithelial degeneration/regeneration of the epithelium in large size bronchi.

 

Trachea

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(7.5)

2

(22.5)

3

(75)

0

(0)

1

(7.5)

2

(22.5)

3

(75)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, epithelial

0

0

0

6

0

0

0

7

·        Grade 1

 

 

 

2

 

 

 

6

·        Grade 2

 

 

 

3

 

 

 

1

·        Grade 3

 

 

 

1

 

 

 

 

 

A multifocal macrovesicular vacuolation of the tracheal epithelium was observed in males and females of test group 3.

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Special stains, performed exemplary in one animal of test group 3 and one control animal were negative for the presence of fat (Oil-Red-O) or glycoproteins (Alcian-blue pH2.5-PAS) in the vacuoles observed in the tracheal and bronchial epithelium of the test group 3 animal.

 

Transmission electron microscopy performed in the same animals revealed in the control tracheal and bronchial epithelium the presence of ciliated and non-ciliated cells. The ciliated cells showed a clear round nuclei and clear cytoplasm with numerous surface cilia. The non-ciliated cells displayed dark nuclei with a characteristic cleft, and cytoplasmic apices bulging into the lumen. The bulging apical side contained electron-lucent single-membrane-bound granules with a slight flocculent content revealing a secretory function. Some of the granules possessed a darker core resembling a “bull-eye“. In the tracheal and bronchial epithelium of the high-dose animal, the non-ciliated cells were most frequently distorted by the presence of very large electron-lucent, well-demarcated spherical vacuoles not bounded by a membrane. The vacuoles size ranged from half the nuclear size to vacuoles that replaced the whole cell cytoplasm strongly compressing the nuclei. The images suggested that the big vacuoles may be derived by fusion of smaller ones. Most of the vacuoles were empty but some of them showed minimal debris most likely cytoplasmic remnants left after the fusion of vacuoles.

Conclusions:
The oral administration of N,N,N´,N´-Tetramethyl-1,6-hexanediamine by gavage to male and female Wistar rats for 3 months caused test substance-related, potential adverse signs of systemic toxicity at a dose level of 75 mg/kg bw/d.
Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 22.5 mg/kg bw/d for male and female Wistar rats.
Executive summary:

N,N,N´,N´-Tetramethyl-1,6-hexanediaminewas administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 7.5 (test group 1), 22.5 (test group 2) and 75 mg/kg bw/d (test group 3) over a period of 3 months.

 

With regard to clinical examinations, no signs of general systemic toxicity were observed in any of the test groups.

 

Regarding clinical pathology decreased absolute and relative eosinophil cell counts indicated some stress reaction of rats of both sexes of test group 3 (75 mg/kg bw/d).

 

Regarding pathology, target organs were the brain, eyes, lungs, and trachea of male and female animals in test group 3 (75 mg/kg bw/d). In all of them, a macrovesicular vacuolation affected epithelial tissues, such as the choroid plexus in the brain, the posterior epithelial layer of the iris in the eyes, the large and medium size bronchioles of the lung and the mucosal epithelium of the trachea. In one male and one female, the vacuolation of the bronchial epithelium was accompanied by a minimal degeneration/ regeneration. Although minimal, these changes revealed the presence of cell injury reflecting at least in the lung an adverse effect.

 

Special stains were negative for the presence of fat or glycoprotein content within the vacuoles observed in the tracheal and bronchial epithelium in the high dose animals. Furthermore, transmission electron microscopy revealed in these epithelia the presence of large round-shaped, electron-lucent vacuoles and excluded the presence oflamellate inclusion bodiescharacteristic of phospholipidosis. Because no further information about a possible pathogenesis of these vacuoles could be reached with these investigations, an adverse effect in the trachea, bronchi, brain and eyes cannot be ruled out.

 

No treatment-related findings were seen in male and female animals of test groups 1 and 2 (7.5 and 22.5 mg/kg bw/d).

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
22.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern screening study (OECD 422) and an Repeated Dose Toxicity Study (OECD 408) are available to address repeated dose toxicity.

Additional information

Effects of treatment in an OECD 422 screening study were limited to reduced eosinophil counts in both sexes at the highest dose level of 75 mg/kg bw/d. Findings in one female at this dose level indicate a possible corrosive effect of the test material on the gastric mucosa.

N,N,N´,N´-Tetramethyl-1,6-hexanediaminewas administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 7.5 (test group 1), 22.5 (test group 2) and 75 mg/kg bw/d (test group 3) over a period of 3 months.

 

With regard to clinical examinations, no signs of general systemic toxicity were observed in any of the test groups.

 

Regarding clinical pathology decreased absolute and relative eosinophil cell counts indicated some stress reaction of rats of both sexes of test group 3 (75 mg/kg bw/d).

 

Regarding pathology, target organs were the brain, eyes, lungs, and trachea of male and female animals in test group 3 (75 mg/kg bw/d). In all of them, a macrovesicular vacuolation affected epithelial tissues, such as the choroid plexus in the brain, the posterior epithelial layer of the iris in the eyes, the large and medium size bronchioles of the lung and the mucosal epithelium of the trachea. In one male and one female, the vacuolation of the bronchial epithelium was accompanied by a minimal degeneration/ regeneration. Although minimal, these changes revealed the presence of cell injury reflecting at least in the lung an adverse effect.

 

Special stainswere negative for the presence of fat or glycoprotein content within the vacuoles observed in the tracheal and bronchial epithelium in the high dose animals. Furthermore,transmission electron microscopy revealed in these epithelia the presence of large round-shaped, electron-lucent vacuoles and excluded the presence oflamellate inclusion bodiescharacteristic of phospholipidosis. Because no further information about a possible pathogenesis of these vacuoles could be reached with these investigations, an adverse effect in the trachea, bronchi, brain and eyes cannot be ruled out.

 

No treatment-related findings were seen in male and female animals of test groups 1 and 2 (7.5 and 22.5 mg/kg bw/d).

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Justification for classification or non-classification

Findings in the available study do not trigger classification of the substance for repeated dose toxicity (STOT-RE) according to current guidance on CLP classification.