Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-05-14 - 2010-09-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid/paste
Details on test material:
- Name of test material (as cited in study report): N-C12,14 alkyl-1,3-diaminopropane
- Physical state: liquid/ paste
- Purity test date: 2007-10-15
- Composition of test material, percentage of components:
N-Alkyl-1,3-diaminopropanes: 94%
N-Alkyl-amines: 6%

Total Primary amines: 5.7 %
Total Diamines: 94.3 %
Iodine number: 2 g I/100g (0-3)
Water: 0.42 % (0-0.5 %)

C12 N Primary amine: 4.3 %
C14 N Primary amine: 1.3 %
C16 N Primary amine: 0.1 %
C12 Diamine: 73.4 %
C14 Diamine: 20.2 %
C16 Diamine: 0.7 %

Acrylnitrile: <4 ppm
Fatty acids: <1 %
Fatty alkyl nitriles: <1 %

- Lot/batch No.: S000904
- Expiration date of the lot/batch: 2010-09-30
- Storage condition of test material: at RT
- pH: basic
- Colour: Max 3 Gardner
- Product name: Duomeen 1214D
- Melting Point: 24-34°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier S.A.S, Route des Chênes secs-B.P.4105-53941 LE GENEST-ST-ISLE-France
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: Main study groups
Females: 177-237 g, (mean: 200.84 g, ± 20%= 160.67 -241.01g)
Males: 246-304 g, (mean: 277.28 g, ± 20%= 221.82- 332.74g)
Recovery groups (28 and 90 day recovery)
Females: 178-218 g, (mean: 196.45 g, ± 20%= 157.16 -235.74g)
Males: 250-289 g, (mean: 267.25 g, ± 20%= 213.80-320.70g)
- Housing: housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in corn oil. The vehicle was chosen due to its non-toxic characteristics as well as according to sponsor`s request. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Lot/batch no. (if required): 117KO127, 058KO 070 and 128KO 040 (Sigma)

Application volume: 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed.
Samples for homogeneity were taken from the top, middle and bottom of the High Dose and Low Dose preparation. The determination was performed in week 1, 5 and 13 of the study. Analysis of the dose formulations of the test item in the vehicle (nominal concentration) was performed in week 1, 5, 9 and 13 of the study for all doses. The dose formulation analysis was performed at BSL Scientific Laboratories GmbH under the BSL Project Nr. 081585.
Duration of treatment / exposure:
up to 90-91 days. The animals in the recovery group were dosed for 90 days and further held for 28 and 90 days post dose treatment period.
Frequency of treatment:
single dose daily, 7 days per week for a period of 90-91 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0.0, 0.1, 0.4, 1.5 and 6 mg/kg bw; Recovery Groups (28 and 90 days): 0 and 6 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 (Main Study); 5 (Recovery Groups)
Control animals:
yes, concurrent vehicle
Details on study design:
Additional information for dosage:
According to the results of the dose range finding study (BSL Study Number 081579) and the 28-Day Repeated Dose Toxicity Study (BSL Study Number 084079) and in consultation with the sponsor the following doses were selected:
Control: 0 mg/kg bw
LD: 0.1 mg/kg bw
MD: 0.4 mg/kg bw
HID: 1.5 mg/kg bw
HD: 6 mg/kg bw
Recovery Groups:
CR: 0 mg/kg bw
HDR: 6 mg/kg bw
The highest dose level was chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and no-observed-adverse effects at the lowest dose level (NOAEL). The animals in the control group were handled in an identical manner to the dose group subjects and received the vehicle in the same volume.



Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection). The observation period was 90-91 days (main study group), 118 days (28-Day recovery group) and 180 days (90-Day recovery group).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made once prior to initiation of the treatment for all animals (main and recovery groups) and weekly thereafter. Once before the first exposure and once in the last week of treatment in all animals (main and recovery groups) and in the 4th week of recovery (28-day recovery groups) and in the last week of recovery (90-day recovery groups), sensory reactivity to stimuli of different types was conducted with specific emphasis on locomotion and behaviour. These observations were made outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight for all animals was measured at randomisation, daily during treatment period and on the day of terminal sacrifice. In recovery animals, after the treatment period, body weight was measured weekly during recovery period.

FOOD CONSUMPTION: Yes
- Food consumption was measured weekly during the treatment period for all animals and weekly during recovery period for recovery animals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once prior to administration of the test article in all animals and once at the terminal sacrifice of main study animals (week 0 and 12).
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples were collected at terminal sacrifice (main and recovery group animals)
- Animals fasted: Yes (overnight)
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: clinical biochemistry determinations were performed on serum samples obtained from all animals as a part of the procedure of killing the animals for necropsy
- Animals fasted: Yes (overnight)
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was performed on the samples collected from main and recovery group animals at terminal sacrifice.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: All animals (main and recovery groups) of the study were subjected to detailed gross necropsy on day 91-92 for main group animals and day 119 and 181 for recovery group animals. During necropsy, a careful examination of the external surface of the body, all orifice and the cranial, thoracic and abdominal cavities and their contents was made. The wet weight of the following organs was taken in all terminally sacrificed animals as soon as possible. Paired organs were weighed separately: liver, ovaries, kidneys, uterus with cervix, adrenal glands, thymus, testes, spleen, epididymides, brain, prostate, heart, and seminal vesicles with coaulating glands

HISTOPATHOLOGY: Yes: The following tissues from all animals were preserved in 10% neutral buffered formalin except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative: all gross lesions, heart, aorta, skin, brain, ovaries, spinal cord, uterus with cervix, eyes, vagina, liver, testes, kidneys, epididymides, adrenal glands, prostate, stomach, seminal vesicles with coaulating glands, small and large intestines, urinary bladder, thymus, lymph nodes, thyroid gland, peripheral nerve, spleen, bone marrow, lungs and trachea, bone and skeletal muscle, pituitary, oesophagus, pancreas, salivary glands, parathyroids, mammary gland, nasal cavity, and larynx.
The afore-listed organs were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Full histopathology was carried out on the preserved organs and tissues of all animals in the control and high dose groups (main groups). These examinations were extended to animals of all other dosage groups as treatment-related changes were observed in the high dose group. All gross lesions were examined histopathologically.
For the recovery groups, histopathology was performed on tissues and organs identified as showing effects in the main groups. Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, GB - Hereford HR2 6JU. Histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.
Other examinations:
Inflammatory Marker
Serum samples collected at terminal sacrifice (main and recovery groups) have been stored at ≤ -20 °C for analysis of inflammatory markers by ELISA technique. The analysis results will be reported separately.
Statistics:
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test or unpaired t test were carried out to reveal any differences between control- and test groups. The statistical analysis was performed with GraphPad Prism V.x software (p < 0.05 was considered as statistical significant).
In the evaluation of laboratory parameters, all values within a range of the mean value ± the two fold standard deviation (x ± 2s) are considered to be „normal“ values within a „normal“ population

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:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Three mortalities in the main groups were observed (2 female HD and 1 male HID). There were also 2 mortalities in high dose females of HDR-90 group.
No test item specific clinical signs were observed in any of the animals of LD and MD of main group. The predominant clinical signs observed in HID and HD main and recovery group animals were slight to severe salivation, half-eyelid closure, vocalization, temporary stertorous respiration, oral and nasal discharge, dyspnoea, weight loss and piloerection.


BODY WEIGHT AND WEIGHT GAIN
In main study males, HD group animals showed statistically significant decrease in overall body weight gain (day 1 - 90) as compared to corresponding controls. Body weight gain values from LD, MD and HID were comparable with that of corresponding control group. In females, all treated group animals showed marginal decrease in overall body weight gain (day 1-90) as compared to corresponding controls. However, this effect was not statistically significant.
In 28 day recovery group males, the HD animals showed a statistically significant decrease in overall body weight gain during the treatment period (day 1-90) as compared to controls. However, body weight gain during 28 day recovery period was increased as compared to controls. In 28 day recovery group female animals, overall body weight gain during the treatment period (day 1 - 90) and 28 day recovery period was comparable to corresponding controls.
In 90 day recovery group males, the HD animals showed a decrease in overall body weight gain during the treatment period (day 1 - 90) as compared to controls without reaching statistical significance. However, body weight gain during 90 day recovery period was increased as compared to controls. In 90 day recovery group female animals, there was no any effect on body weight during treatment and recovery period.
The difference in weight gain among the groups could be attributed to the treatment with test item.


FOOD CONSUMPTION
In main study males, HD group animals showed statistically significant decrease in food consumption (day 1 - 90) as compared to corresponding controls. Food consumption from LD, MD and HID was slightly decreased without reaching statistical significance when compared with that of corresponding control group. In females, all treated group animals showed decrease in food consumption (day 1 - 90) as compared to corresponding controls. However, this effect was not statistically significant. In 28 day recovery group males, the HD animals showed a decrease in food consumption during the treatment period (day 1 - 90) as compared to controls without achieving statistical significance. However, food consumption during 28 day recovery period was slightly increased as compared to controls. In 28 day recovery group female animals, food consumption during the treatment period (day 1 - 90) and 28 day recovery period was comparable to corresponding controls. In 90 day recovery group males, the HD animals showed a decrease in food consumption during the treatment period (day 1 -90) as compared to controls without reaching statistical significance. However, food consumption during 90 day recovery period was increased as compared to controls. In 90 day recovery group female animals, there was no significant effect on food consumption during treatment and recovery period. The difference in food consumption among the groups could be attributed to the treatment with test item as it was correlated with body weight decrease during the respective period.


HAEMATOLOGY
The blood analysis for haematology and clinical pathology parameters revealed most of the mean and individual values within the biological range. Statistically significant differences in few parameters were found between the treated groups and the corresponding control group.
For hematology, except for Hct values in MD and HID males and MD, HID and HD females, RBC in main study males of MD group and females of HDR-28, MCV in HDR-28 females, MCH in HDR-28 females, MCHC in MD and HDR- 28 males, WBC in HD males, no other parameters showed any significant difference compared to controls. Individual values for most of the parameters were within the biological range.
In most of the main and recovery group males and females, the mean and individual PTT and aPTT values were below the biological range. Statistically significant decrease in HD main study males were observed for PTT when compared to the controls.
Statistically significant decrease in aPTT was observed in LD, MD, HID males, LD, MD females and HDR-90 males when compared with respective controls.


CLINICAL CHEMISTRY
For clinical biochemistry, except for AST/GOT in HD males, TP in female HID and HD group, GLU in HDR-90 males, UREA in female HID, no other parameters showed any significant deviation compared to controls. No treatment related effect on urine parameters were found in treated groups as compared to control group.


URINALYSIS
No relevant differences between test and control groups were found for both main and recovery animals. No dose dependency and toxicological relevance was observed in various urine parameters.
Low pH values (acidic urine, pH 5-6) were observed in few animals.
Main study groups: 6/10 male and 9/10 female control; 6/10 male and 7/10 female of LD group; 2/10 male and 7/10 female MD group; 5/10 male and 6/10 female HID group; 10/10 male and 8/8 female HD group.
28 day recovery groups: 2/5 male and 4/5 female control; 5/5 male and 3/3 female HD group.
90 day recovery groups: 2/5 male and 3/5 female control; 2/5 male and 3/3 female HD group.


ORGAN WEIGHTS
No effect on organ weights was observed except statistically significant decrease in absolute liver weight in HD males, increase in relative kidney weights in HDR-28 males, increase in relative adrenal weights in male and female HD, increase in relative heart weights in males of MD group, decrease in absolute brain weights in HID males, increase in relative brain weights in HD males and decrease in absolute brain weights in HDR-28 males and increase in relative epididymides weights in HD males.


GROSS PATHOLOGY
At terminal sacrifice of main study and recovery animals, macroscopic findings observed were considered to be spontaneous and not treatment related.


HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related changes were seen in the small intestine (ileum and jejunum), mesenteric lymph node, spleen, bone marrow (sternum) and trachea of terminally sacrificed animals.
After the 28-day recovery period, in animals of the HDR-28 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node were still present. Changes noted in the spleen had partially regressed and bone marrow (sternum) and trachea changes had fully regressed.
After 90 days of recovery, in animals of the HDR-90 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node had partially regressed. No test item-related changes were noted in the spleen, bone marrow (sternum) and trachea.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
0.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Haematology data

Group

Parameter

Hct

RBC

MCV

MCH

MCHC

WBC

PTT

aPTT

Unit

%

x106/µl

fL

pg

g/dL

x103/µl

sec

sec

Biol. Range

28.3-54.9

6.3-10.54

45.5-60.8

15.4-19.8

30.7-36.7

2.9-25

29-51

10.9-37

Male

C

Mean

SD

N

49.05

1.5

10

9.07

0.37

10

54.11

1.55

10

17.03

0.71

10

31.5

0.6

10

10.63

2.06

10

27.61

5.36

10

10.69

3.44

10

LD

Mean

SD

N

50.95

3.47

10

9.33

0.64

10

54.63

1.51

10

17.07

0.6

10

31.24

0.59

10

10.53

3.05

10

20.61*

5.06

10

11.28

2.34

10

MD

Mean

SD

N

53.72*

2.53

8

9.83*

0.5

8

54.66

0.76

8

16.73

0.35

8

30.58*

0.61

8

10.01

0.74

8

19.98*

4.43

8

10.95

2.12

8

HID

Mean

SD

N

52.87*

4.11

9

9.59

0.76

9

55.15

1.03

9

17.06

0.49

9

30.92

0.58

9

9.33

2.36

9

17.32*

3.2

9

9.75

1.37

9

HD

Mean

SD

N

49.71

2.78

10

9.31

0.36

10

53.37

1.49

10

16.74

0.63

10

31.37

0.83

10

16.19*

6.77

10

25.44

6.42

10

11.35

2.22

10

CR28

Mean

SD

N

48.96

2.63

5

9.01

0.65

5

54.44

2.14

5

17.58

0.82

5

32.29

0.54

5

11.26

2.05

5

21.12

1.95

5

11.49

0.88

5

HDR28

Mean

SD

N

52.99

3.39

5

9.94

0.71

5

53.33

1.34

5

16.54

0.68

5

31.01*

0.81

5

13.32

3.06

5

23.39

3.16

5

12.26

2.58

5

CR90

Mean

SD

N

48.69

2.0

5

8.74

0.57

5

55.76

1.92

5

18.06

1.07

5

32.4

0.84

5

9.46

1.92

5

45.99

4.67

5

15.19

2.41

5

HDR90

Mean

SD

N

49.49

2.28

5

8.98

0.41

5

55.11

1.04

5

17.47

0.51

5

31.69

0.77

5

10.76

3.54

5

34.28*

4.11

5

13.1

0.41

5

Female

C

Mean

SD

N

43.92

1.32

10

7.93

0.41

10

55.47

1.29

10

18.12

0.7

10

32.66

0.8

10

5.46

2.22

10

22.4

11.9

10

11.15

3.73

10

LD

Mean

SD

N

45.58

1.5

10

8.28

0.27

10

55.07

0.36

10

18.0

0.45

10

32.69

0.78

10

5.59

0.84

10

13.6*

5.38

10

8.66

3.1

10

MD

Mean

SD

N

46.84*

2.46

10

8.42

0.4

10

55.62

1.74

10

17.84

0.43

10

32.08

0.53

10

4.96

1.2

10

14.51*

1.23

10

9.31

1.39

10

HID

Mean

SD

N

46.35*

2.83

10

8.37

0.51

10

55.41

0.81

10

17.7

0.37

10

31.94

0.7

10

5.17

2.28

10

16.9

2.77

10

9.65

1.12

10

HD

Mean

SD

N

46.66*

1.08

8

8.38

0.33

8

55.71

1.74

8

17.9

0.44

8

32.14

0.58

8

7.44

2.38

8

19.11

4.44

8

10.49

1.44

8

CR28

Mean

SD

N

45.89

1.24

5

8.14

0.1

5

56.34

0.94

5

18.48

0.57

5

32.76

0.6

5

4.39

2.26

5

24.88

5.23

5

12.91

1.5

5

HDR28

Mean

SD

N

46.46

1.72

5

8.52*

0.3

5

54.5*

0.41

5

17.66*

0.5

5

32.36

0.82

5

5.32

1.77

5

25.24

2.45

5

12.44

1.05

5

Table 2: Clinical biochemistry data

Group

Parameter

AST/GOT

TP

GLU

UREA

Unit

U/L

g/L

mmol/L

mmol/L

Male

C

Mean

SD

N

71.39

73.45

10

55.85

2.93

10

13.17

2.63

10

6.0

0.95

10

HD

Mean

SD

N

40.23

12.91

10

53.25

4.56

10

13.05

1.03

10

6.81

0.84

10

CR90

Mean

SD

N

44.36

3.09

5

59.34

0.97

5

14.74

1.23

5

6.91

0.65

5

HDR90

Mean

SD

N

139.02

193.73

5

57.66

1.35

5

12.23*

1.76

5

7.14

1.02

5

Female

C

Mean

SD

N

41.48

11.35

9

58.81

4.48

9

8.89

1.2

9

6.78

0.46

9

HID

Mean

SD

N

30.62

4.42

10

55.21*

2.96

10

9.88

2.48

10

5.57*

0.79

10

HD

Mean

SD

N

33.63

6.1

8

53.08*

2.55

8

9.92

1.25

8

6.01

1.11

8

*Significant (p<0.05); as determined with the individual data

Applicant's summary and conclusion

Conclusions:
Test item related clinical signs were observed in HID and HD main groups and HD recovery groups. Predominant signs observed were reduced spontaneous activity, salivation, nasal discharge and moving the bedding.

Three mortalities in the main groups, (two female from HD and one male from HID) and 2 mortalities in high dose females of HDR-90 group were observed.

In main study and recovery HD group male and female animals, there was statistically significant decrease in overall body weight gain (day 1-90) and food consumption was observed compared to corresponding controls. Body weight gain and food consumption values from MD and HID were slightly decreased without achieving a statistical significance when compared with control. However, these parameters remained unaffected in low dose and values were comparable with the controls.

No test item related effect in any of the haematology parameters, urine parameters and clinical biochemistry parameters in male and females of any group except decrease in TP in HD females was observed.

At terminal sacrifice of main study and recovery animals, macroscopic findings observed were considered to be spontaneous and not treatment related

No effect on organ weight in male and female animals of treated group and recovery group was observed which could be considered as a treatment related effect except decrease in absolute liver weights in HD males, increase in relative kidney weights in HDR-28 males, increase in relative adrenal weights in HD male and females and increase in relative epididymides weight in HD males.

Histopathologically, test item-related changes were seen in the small intestine (ileum and jejunum), mesenteric lymph node, spleen, bone marrow (sternum) and trachea of the MD, HID and HD animals. After the 28-day recovery period, item-related changes in the jejunum, ileum and mesenteric lymph node were still present in animals of the HDR-28 dose group. Changes noted in the spleen had partially regressed and bone marrow (sternum) and trachea changes had fully regressed.
After 90 days of recovery, in animals of the HDR-90 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node had partially regressed.
No test item-related changes were noted in the spleen, bone marrow (sternum) and trachea.
All findings observed in this 90 day study are comparable with 28 day repeated dose toxicity study performed with N-C12,14 alkyl-1,3-diaminopropane (BSL Study No. 084079). It is indicative that the observed effects are local and might have induced by inflammatory changes and therefore treatment related observed effects in various parameters could be secondary changes to the inflammatory responses.
This 90 day repeated dose oral toxicity study including a 28 and 90 day recovery period revealed treatment related changes in few parameters of high dose group male and females. Except histopathology, slight effect on body weight, food Report BSL consumption and clinical signs, no test item related effect was observed in any of the parameters evaluated in MD and HID group. Based on these findings and the histopathological evaluation, the NOAEL (No-Observed-Adverse-Effect-Level) was considered to be 0.4 mg/kg/day under the circumstances of this study.
Executive summary:

In this 90 day repeated dose oral toxicity study (performed according to the First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 407, “Repeated Dose 28-day Oral Toxicity Study in Rodents” adopted 03 October 2008, First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 408, “Repeated Dose 90 day Oral Toxicity Study in Rodents” adopted 21 September 1998, Directive 96/54 EEC B.7 and Directive 2001/59/ EC B.26), the test item N-C12,14 alkyl-1,3-diaminopropane was suspended in corn oil and orally administered in graduated doses to four groups of male and female rats (Wistar RjHan:WI) by oral gavage, using a gavaging canula. The main study included 5 groups (C, LD, MD, HID, HD) with each comprised of 10 male and 10 female animals. 28 day and 90 day recovery study included 2 groups (CR-28, HDR-28, CR-90 and HDR-90) each with 5 male and 5 female animals. 

Total 90 applications per animal were administered and the animals of the recovery groups were kept for further 28 and 90 days following the last administration before terminal sacrifice. 

The following doses were chosen: Low Dose: 0.1 mg/kg bw, Medium Dose: 0.4 mg/kg bw, High Intermediate 1.5 mg/kg bw and High Dose: 6 mg/kg bw. The volume of application was 4 mL/kg bw.

All surviving animals were sacrificed on day 91 – 92 (main group animals) day 119 and 181 (recovery groups). 

Clinical signs and Mortality

No test item specific clinical signs were observed in any of the animals of control LD, MD of main group. The predominant clinical signs observed in HID and HD main and recovery group animals were slight to severe salivation, half-eyelid closure, vocalization , temporary stertorous respiration, oral and nasal discharge, dyspnoea, weight loss and piloerection.

Three mortalities in the main groups were observed ( two female HD and one male HID).There were also 2 mortalities in high dose females of HDR-90 group.

 

Body Weight

In main study males, HD group animals showed statistically significant decrease in overall body weight gain (day 1-90) as compared to corresponding controls. In females, all treated group animals showed marginal decrease in overall body weight gain (day 1-90) as compared to corresponding controls. However, this effect was neither dose related nor statistically significant.

In 28 day recovery group males, the HD animals showed a statistically significant decrease in overall body weight gain during the treatment period (day 1-90) as compared to controls. However, body weight gain in during 28 day recovery period was increased as compared to controls. In 28 day recovery group female animals, overall body weight gain during the treatment period (day 1-90) and 28 day recovery period was comparable to corresponding controls. 

In 90 day recovery group males, the HD animals showed a decrease in overall body weight gain during the treatment period (day 1-90) as compared to controls without reaching statistical significance. However, body weight gain during 90 day recovery period was increased as compared to controls. In 90 day recovery group female animals, there was no any effect on body weight during treatment and recovery period.

 

Food Consumption

In main study males, HD group animals showed statistically significant decrease in food consumption (day 1-90) as compared to corresponding controls. Food consumption from LD, MD and HID was slightly decreased without reaching statistical significance when compared with that of corresponding control group. 

In females, all treated group animals showed decrease in food consumption (day 1-

90) as compared to corresponding controls. However, this effect was not statistically significant. 

In 28 day recovery group males, the HD animals showed a decrease in food consumption during the treatment period (day 1-90) as compared to controls without achieving statistical significance. However, Food consumption during 28 day recovery period was slightly increased as compared to controls. In 28 day recovery group female animals, food consumption during the treatment period (day 1-90) and 28 day recovery period was comparable with corresponding controls. In 90 day recovery group males, the HD animals showed a decrease in food consumption during the treatment period (day 1-90) as compared to controls without reaching statistical significance. However, food consumption during 90 day recovery period was increased as compared to controls. In 90 day recovery group female animals, there was no significant effect on food consumption during treatment and recovery period.  

 

FOB

No differences were observed concerning functional and behavioral examination prior to application and during the last week of treatment. No treatment related abnormalities were recorded concerning posture, gait, palpebral closure, lacrimation, piloerection, arousal and vocalization. No convulsions, tremors, stereotypy or bizarre behaviour were observed in any of the animals.

 

Haematology and Clinical Biochemistry

The blood analysis for haematology and clinical pathology parameters revealed most of the mean and individual values within the biological range. Statistically significant differences in few parameters were found between the treated groups and the corresponding control group. 

 

For hematology, except for Hct values in MD and HID males and MD, HID and HD females, RBC in in main study males of MD group and females of HDR-28, MCV in in HDR-28 females, MCH in HDR-28 females, MCHC in MD and HDR- 28 males, WBC in HD males, no other parameters showed any significant difference compared to controls. Individual values for most of the parameters were within the biological range.

 

Effect on clotting parameters like PTT and aPTT can not be attributed to the treatment with test item and considered to be an incidental due to lack of dose dependency and majority of the group mean values were within the biological range. 

 

Statistically significant decrease in aPTT was observed in LD, MD, HID males, LD, MD females and HDR-90 males when compared with respective controls. 

 

For clinical biochemistry, except for AST/GOT in HD males, TP in female HID and HD group, GLU in HDR-90 males, UREA in female HID, no other parameters showed any significant deviation compared to controls.   

No treatment related effect on urine parameters were found in treated groups as compared to control group.

 

Gross Pathology

At terminal sacrifice of main study and recovery animals, macroscopic findings observed were considered to be spontaneous and not treatment related.

 

Organ Weight

No effect on organ weights was observed except statistically significant decrease in absolute liver weight in HD males, increase in relative kidney weights in HDR-28 males, increase in relative adrenal weights in male and female HD, increase in relative heart weights in males of MD group, decrease in absolute brain weights in HID males, increase in relative brain weights in HD males and decrease in absolute brain weights in HDR-28 males and increase in relative epididymides weights in HD males.

 

Histopathology

Test item-related changes were seen in the small intestine (ileum and jejunum), mesenteric lymph node, spleen, bone marrow (sternum) and trachea of terminally sacrificed animals.

 

After the 28-day recovery period, in animals of the HDR-28 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node were still present. Changes noted in the spleen had partially regressed and bone marrow (sternum) and trachea changes had fully regressed.

 

After 90 days of recovery, in animals of the HDR-90 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node had partially regressed. No test item-related changes were noted in the spleen, bone marrow (sternum) and trachea.