Registration Dossier

Diss Factsheets

Administrative data

Description of key information

A 28-day study on Oleyl-diamine dioleate resulted to a NOAEL of 5 mg/kg bw/day is available.  A read-across to diamines category is applied taking into account a 28-day study with Oleyl-diamine (NOAEL of 1.25 mg/kg bw/day) and a 90-day study with N-C12-14-diamine( NOAEL of 0.4 mg/kg bw/day equivalent to 1.1 mg Oleyl-diamine dioleate/kg bw/day).





Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-05-14 - 2010-07-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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
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 days
Frequency of treatment:
single dose daily, 7 days per week for a period of 90-91 days
Remarks:
Doses / Concentrations:
0.0, 0.1, 0.4, 1.5 and 6 mg/kg bw; Recovery Group: 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
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)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to initiation of the treatment for all animals (main and recovery groups) and weekly thereafter.

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:
- 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.
- 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
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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.
Mortality:
mortality observed, treatment-related
Description (incidence):
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.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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.
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
Description (incidence and severity):
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 biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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 findings:
effects observed, treatment-related
Description (incidence and severity):
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.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No differences were observed concerning functional and behavioural examination prior to application and during the last week of dosing in main and recovery animals.
No abnormalities were recorded concerning posture, gait, palpebral closure, lacrimation, piloerection, arousal and vocalisation.
No convulsions, tremors, stereotypy or bizarre behaviour were observed in any of the animals of main and recovery groups.
For supported and unassisted rears no abnormalities were detected. Responses to reflex testing were normal in all groups.
On measures such as counts of urination and defecation differences could not be detected.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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 pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At terminal sacrifice of main study and recovery animals, macroscopic findings observed were considered to be spontaneous and not treatment related.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
not examined
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
other: At 1.5 mg/kg bw/day, changes in the jejunum, ileum and mesenteric lymph node had partially regressed after a 90-day recovery period.
Critical effects observed:
not specified

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

Conclusions:
This 90 day repeated dose oral toxicity study including a 28 and 90 day recovery period revealed treatment related changes. 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). 


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


Clinical signs


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.


 


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 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 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 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.


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. 


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.


 


 


The NAOEL of 0.4 mg/kg for C12-14-diamine is taken in cross-reading as the NOAEL value for Oleyl-diamine, which then is converted to the corresponding value for the dioleate salt of Oleyl-diamine.


(mw oleyl-diamine = 324.7, mw oleyl-diamine dioleate = 889.5; 0.4 x 889.5/324.7 = 1.1 mg/kg bw/day)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality studies, showing consistent results and justifying the cross-reading from the group of diamines for the use of a 90-day study (OECD 408) to cover Annex IX requirements. Studies are GLP compliant with Klimisch score 1. The applied read-across is adequate and likely to result to an overestimation of toxicity as testing is performed on the most toxic member of the diamine category. The NOAEL is based on cross-reading from 0.4 mg/kg of C12-14-diamine as NOAEL value for Oleyl-diamine, which then is converted to the corresponding value for the dioleate salt of Oleyl-diamine.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral route:


Oleyl-diamine dioleate is a salt of Oleyl-diamine and oleic acid, in a molar ratio of 1:2. The hypothesis is that the salt Oleyl-diamine dioleate will after absorption dissociate, and that therefore its toxicity would principally be based on its Oleyl-diamine content (based on the much more severe toxicity profile of alkyl-diamines compared to fatty acids).


Therefore, it was decided to perform a 28-day study on Oleyl-diamine dioleate, and compare the results with those of the 28-day study of Oleyl-diamine.


 


 


Oleyl-diamine dioleate - 28-day


The repeated dose toxicity of Oleyl-diamine dioleate was evaluated in 28-day oral gavage study in rats according to OECD 407 guideline and in compliance to GLP.


The selected dose levels were based on the results of a range finding/MTD study.


Four groups of 5 male and 5 female Wistar rats received Oleyl-diamine dioleate dissolved in corn oil at dose levels of 0, 5, 20 and 100 mg/kg bw/day. Formulations were chemically analysed.


The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.


Effects at 100 mg: Clinical signs consisted of hunched posture, piloerection and lean appearance and lower motor activity. Body weights were lower than control (male 21%, females 14%) and at the clinical laboratory examinations WBC (neutrophils counts) was increased and albumin was decreased. Macroscopic examinations showed thickening and dilation of the jejunum, and reddish discolouration and enlargement of the mesenteric lymph nodes. Histological correlates of these findings were foamy macrophages foci present in the lamina propria of the duodenum, jejunum and/or ileum of both sexes. Moreover, (foamy) macrophage foci, granulocytic cell infiltrate and/or cellular debris, granuloma, granuloma with central necrosis, and lymphoid hyperplasia were noted in the mesenteric lymph nodes of male and female rats.


In general, macrophage foci present in the mesenteric lymph nodes may develop into more vacuolated (foamy) macrophages when taking up more material and finally being organized at higher doses into granuloma. In addition, granulocytes and cellular debris were present in these affected lymph nodes and in males some granuloma developed central necrosis.


At 20 mg/kg, foamy macrophage foci in the lamina propria of the jejunum in one female were observed. In addition, in the majority of the rats (foamy) macrophage foci, granulocytic cell infiltrate and/or cellular debris, granuloma and lymphoid hyperplasia were noted in the mesenteric lymph nodes.


At 5 mg/kg, foamy macrophage foci in the mesenteric lymph nodes was noted in one female only at a minimal degree. Given the incidental and minor nature of this finding, this was considered of no toxicological relevance.


Based on these results, a No Observed Adverse Effect Level (NOAEL) was established at the lowest dose level of 5 mg/kg.


 


Oleyl-diamine - 28-day:


Oleyl-diamine (CAS 7173-62-8) has been evaluated for repeated dose toxicity in a 28-day repeated dose toxicity study according to OECD 407 guideline and in compliance to GLP. Four groups of five male and female rats were exposed by gavage to dose levels of 0, 1.25, 5, 20 mg Oleyl-diamine in corn oil/kg bw.


An additional group of control and high dose animals were subjected to the same treatment but with a 14 day recovery.


In the high dose group there was one substance related death (prominent enteropathy of the small intestine with villous infiltration with foam cells and foam cells in mesenteric lymph nodes.). Further observations in the 20 mg/kg group were decreased BW gain, clinical signs (vocalization, piloerection, salivation, arched back, half eyelid closure, sneezing, reduced/increased spontaneous activity, diarrhea and dyspnoea.), increased WBC (neutrophils) and decreased albumin. Furthermore organ weights (absolute and relative) for brain, heart, thymus, testes and prostate showed differences to controls. All these effects had regressed in the recovery groups.


At 5 mg/kg clinical signs were observed of vocalization, increased spontaneous activity, nervousness and piloerection. Haematology showed increased WBC (neutrophils) and biochemistry mainly decreased albumin.


No test item specific clinical signs were observed in any of the animals of control and 1.25 mg/kg group


Histopathological examinations showed foam cell accumulations in small intestinal villi and mesenteric lymph node with accompanying inflammatory changes in small intestine, mesenteric lymph node and spleen essentially at 5 and 20 mg/kg/day, tracheal epithelial alteration and (multi)focal myocardial degeneration/fibrosis of the heart at 5 and 20 mg/kg/day, increased myeloid:erythroid ratio in the bone marrow at 20 mg/kg/day and foam cell accumulation in the thymus in a single animal at 20 mg/kg/day. Minimal foam cell accumulations in small intestinal villi were also observed in a single female at 1.25 mg/kg/day. After the recovery period, in animals of the 20 mg/kg/day dose group, foam cell accumulations in the small intestine and mesenteric lymph node had not regressed. Inflammatory intestinal, mesenteric lymph node and spleen changes had partially regressed.


The NOAEL was considered to be 1.25 mg/kg/day under the circumstances of this study.


 


 


The 28-day study results of Oleyl-diamine dioleate form an excellent match with the results from the 28-day on Oleyl-diamine. This makes clear that the toxicological profile of Oleyl-diamine dioleate is fully determined by its Oleyl diamine content. The 28-day study on Oleyl-diamine resulted to a NOAEL 1.25 mg/kg bw/day (equal to 3.4 mg/kg bw/day as Oleyl-diamine dioleate on the basis of diamine content considered as Oleyl diamine).


 


Further evaluation for repeated dose toxicity of Oleyl-diamine involving sub-chronic studies is based on read-across from C12-14-diamine (N-C12-/4 alkyl-1,3-diaminopropane, CAS 90640-43-0). Within the category of alkyl-diamines the spectrum of toxic effects is similar between the category members, but the level of toxicity is related to the chain length, with the shorter chain-length resulting to a lower NOAEL compared to a diamine based on a longer chain-length. Therefore, within the category of diamines, data on C12-14-diamine can be regarded as representative for the assessment of the whole category, and a worst case approach in case the diamine is based on a longer alkyl chain such as is the case for Oleyl-diamine.


 


 


C12-14-diamine 28-day & 90-day:


The evaluation of the repeated toxicity of C12-14-diamine is based on a 28 day and 90 day study performed according to OECD guideline and under GLP:


 


The 28 day study was performed at dose levels of 0, 0.4, 1.5 and 6 mg/kg bw/day administered per gavage for 28 days. An additional group of animals were subjected to the same treatment but with a 14 day recovery. Relatively minor changes in haematological, i.e. increase in neutrophiles and decrease in lymphocytes, and clinical chemistry (total protein) parameters were observed in the high dose group. The finding of the most significant toxicological relevance is the histopathological changes in small intestine and mesenteric lymph nodes which indicated accumulation of foam cells. These changes regressed, but were still present, following the 14 day recovery period. A NOAEL of 0.4 mg/kg bw/day was established based on these findings.


 


The 90 day study was performed with dose groups of 0, 0.1, 0.4, 1.5 and 6 mg/kg bw/day (in corn oil) administered per gavage daily for 90 days. Additional groups of animals were administered 0 or 6 mg/kg bw/day for 90 days and were then observed for recovery over 28 or 90 days. There was mortality in the highest dose group but some of the deaths are attributed to gavaging errors. However, it cannot be excluded that substance-related mortality was observed in the highest dose group. There were no effects in the two lowest dose groups and the NOAEL was set at 0.4 mg/kg bw/day.


The main effects in highest dose group were on bodyweight gain, with recovery following end of dosing, and organ weights, such as liver, kidney, adrenal glands and epidydimis. These changes were not accompanied by histological changes and they were specific to one sex, with the exception of adrenal gland. The toxicological relevance of these findings can be questioned. There were, however, other substance-related histological changes on the small intestine, i.e. the ileum and jejunum, mesenteric lymph nodes, spleen, bone marrow and trachea. The histological changes in both the intestine and mesenteric lymph nodes were indicative of accumulation and infiltration of foam cells. The changes in the small intestine did show regression during the recovery period, but were still present after 90 days.


 


Evaluation:


The most significant treatment-related changes in all studies are increased WBC (neutrophils) at higher dose levels combined with changes in body weight gain. The critical effects are histopathologically, test item-related effects on the small intestine and mesenteric lymph nodes, which only partially regress during the recovery period. A relatively strong inflammatory reaction is also observed. These effects have consistently been observed with these sort of substances (Fatty amine derived substances), which support the current approach of grouping of substances and read-across of data. When taking into consideration the relatively strong corrosive effects of these substances and the route of administration, it cannot be excluded that the overall toxicity reflects a point-of-first-contact effect. A mode of action has not been established but it is possible to suspect the known corrosivity to be at least partially involved.


The observed effects of foamy macrophages are local and are by some interpreted as phospholipidosis (PLDsis), something which is commonly observed following treatment with cationic amphiphilic drugs and considered to be non-adverse.


 


 


When comparing the 28-day results on Oleyl-diamine dioleate with the 28-day study on Oleyl diamine clearly shows a comparable toxicological profile.For both studies the effects observed, as well as the levels of the effects are very similar resulting to the same NOAEL when based on Oleyl-diamine content: 1.8 mg Oleyl-diamine/kg bw for Oleyl-diamine dioleate (based on NOAEL of 5 mg/kg bw/day for Oleyl-diamine dioleate itself) and 1.25 for Oleyl-diamine (differences are related to dose selection per study rather than actual differences in response).


Also the further studies on C12-14-diamine show the same toxicological responses.


The results from C12-14-diamine are considered to represent a worst case situation for Oleyl-diamine (and thus also for Oleyl-diamine dioleate),as both substances share the same molecular structure, but due to its overall smaller alkyl chain length, its solubility and potential to pass cell-membranes is expected to be possibly somewhat higher than that of Oleyl-diamine. Therefore, any inherent hazards for systemic toxicity are more likely to be expressed when testing with C12-14-diamine than with Oleyl-diamine.


 


All findings observed in the 90-day repeated dose toxicity study are comparable with the 28-day repeated dose toxicity study performed with C12-14-diamine, which indicates that duration has no great impact on the NOAEL.


Small intestinal and mesenteric lymph node lesions show only partial and slowly reversibility. The effects were not reversible during the 28-day recovery period, but had partially regressed after the 90-day recovery period. Other effects seen at higher dose levels as spleen, bone marrow and tracheal changes had completely regressed after the 28-day and 90-day recovery period.


 


 


In conclusion:


The above data shows that 28-day studies on Oleyl-diamine dioleate and Oleyl-diamine show the same toxicological profile and NOAEL when based on Oleyl-diamine content. This indicates the possibility of cross-reading from alkyl-diamines to the alkyl-diamine dioleates. The data on C12-14-diamine can be taken as representative for the alkyl-diamines, and the 90-day NOAEL can be taken considered a worst case approach for the alkyl-diamines with higher alkyl chain-lengths than C12-14.


Taking the 90-day NOAEL of 0.4 mg/kg bw for C12-14-diamine as NOAEL for Oleyl-diamine (as worst case), this would equal to a NOAEL of 1 mg/kg bw on the basis of Oleyl-diamine dioleate. This value is taken for the starting point for DNEL calculations for Oleyl-diamine dioleate (mw oleyl-diamine = 324.7, mw oleyl-diamine dioleate = 889.5; NOAEL 0.4 mg/kgbw x 889.5/324.7 = 1.1 mg/kg bw).


 


An additional observation is that for risk assessment derivation of DNELs for Oleyl-diamine dioleate, the DNEL based on 90-day (sub-chronic) study on C12-14-diamine (NOAEL 1 mg/kg calculated as Oleyl-diamine dioleate) is lower than the DNEL when based on the NOAEL from the 28-day (sub-acute) study on Oleyl-diamine dioleate itself (NOAEL 5 mg/kg bw). This because the extrapolation factor from sub-acute to sub-chronic of a factor 3 leads to a value of 1.67 mg/kg bw.


The data indicates that duration of the study has no great impact on the NOAEL. This lower value can therefore be considered as an extra level of conservatism in the risk assessment.


 


Dermal route:


No data is available for dermal route. The skin is not a preferred route when studying repeated dose systemic toxicity.


 


Inhalation route:


Oleyl-diamine dioleate is a very viscous fluid and has a very low vapour pressure (< 0.0015 Pa at 20°C based on C12/14-diamine rather than Oleyl diamine dioleate salt) and the indicated use patterns also does not give rise to a high concern for specific exposures via inhalation of this oleyl-diamine dioleate salt. Due to its very low vp, any relevant exposure can only be in the form of aerosol. As the use does not include fine spraying, in can in practice only concern larger droplets. Droplets will deposit mainly on upper airways, and will be subsequently swallowed following mucociliary transportation to pharynx. This results to no principal difference in absorption compared oral route. Furthermore, the substance is irritating, and effects are likely to be dominated by local irritation rather than lead to significant systemic uptake.

Justification for classification or non-classification

STOT-RE Cat,1 is required in case of significant toxicity at levels = 10 mg/kgbw/d in 90-day studies or = 30 mg/kgbw/d in case of 28-day studies. For Cat.2 classification levels up to 10 times higher apply.

 

The available 28-day study on Oleyl-diamine dioleate resulted to a NOAEL of 5 mg/kgbw. The highest tested dose of 100 mg/kg results possibly not to sufficient for severe toxicity, although indications of disseminated inflammatory response with increased WBC all animals and myeloid hyperplasia in the sternal bone marrow in 3/5 males were reported. A range finding study with 400 mg/kg was too toxic within 10 days. These data are not sufficient for classification STOT-RE 2 (severe effects below 300 mg/kg), but does not rule out possibility.

 

The data on Oleyl-diamine dioleate fully agrees to cross-reading to diamines (Fraction of oleyl-diamine is 36.5%). Consequently cross-reading to the full repeated dose endpoint data of the diamines will be done:

The available data on Oleyl-diamine, which in turn also includes cross-reading to C12/14-diamine as only diamine for which a 90-day study is available, indicates a classification Cat.1 for STOT-RE:

- 90-day study with N-C12-14-diamine: This study showed significant toxicity at the highest tested dose of 6 mg/kgbw/day based on mortality observed in the highest dose group. This is below 10 mg/kg bw and therefore also results to classification STOT-RE Cat.1.

- 28-day study with Oleyl-diamine: This resulted to significant toxicity (mortality) at the highest dose of 20 mg/kgbw/day with effects on weight of body and organs without histological changes. This is below 30 mg/kg bw and therefore also results to classification STOT-RE Cat.1.

 

When oleyl-diamine leads to mortality at 20 mg/kgbw, a mixture containing 20% or more is expected to lead to severe toxicity at =100 mg/kg, and this would lead to STOT-RE Cat.2. Although this was not fully supported in the actual test with Oleyl-diamine dioleate, it is prudent to classify this substance as STOT-RE Cat. 2.H373. Effects are based on “morbidity or death”.