Registration Dossier

Administrative data

Description of key information

Oral (OECD 408): NOAEL (subchronic) >= 1000 mg/kg bw/day (male/female)
Waiving - Repeated dose toxicity: inhalation
Waiving - Repeated dose toxicity: dermal

Key value for chemical safety assessment

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:
27 Aug 2014 - 13 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP with an additional focus on the effects on reproductive tissues and organs and fertility parameters.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(1998)
Deviations:
yes
Remarks:
additional focus on fertility parameters
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Limit test:
no
Species:
rat
Strain:
other: Wistar rats, Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 - 7 weeks (males/females)
- Weight at study initiation: 113 - 140 g (females), 146 - 180 g (males)
- Housing: The animals were housed in groups (5 animals/sex/cage) in type IV polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1526), ad libitum
- Water: Sulphur acidified tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 Sep 2014 To: 09 Dec 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was ground to a fine powder with the help of a mortar and pestle. The test item was weighed into a tarred plastic vial on a precision balance and the vehicle was added to give the appropriate final concentration of the test item. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test item’s characteristics.
- Concentration in vehicle: 14.29 mg/mL (low dose, LD), 42.86 mg/mL (medium dose, MD), 142.86 mg/mL (high dose, HD)
- Amount of vehicle (if gavage): 7 mL/kg bw/day
- Lot/batch no. (if required): MKBP7039V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration analysis of formulation samples was determined in study weeks 1, 5, 9 and 13 for all dose groups. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 93.3%, 98.8% and 99.8% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentration did not differ from nominal concentration by more than the acceptance criterion of 20%. Stability of formulation samples was investigated in study week 1 based on concentration in the LD, MD and HD dose groups. After 3 hours storage at room temperature recovery compared to starting value was between 110.0% and 118.4%. All samples were stable, as concentration after storage did not differ from the start value by more than the acceptance criterion of 20%. Homogeneity of formulation samples was determined in study weeks 1, 5 and 13 for the LD, MD and HD dose groups. The mean recovery observed for the LD dose group was 104.5%, 100.4% and 95.2% of the nominal value, for MD dose group 101.7%, 109.3% and 103.0% of the nominal value and 107.3%, 111.3% and 108.3% of the nominal value for HD dose group. The coefficients of variation (COV) of the different sampling locations (top, middle,bottom) were 10.0%, 9.3% and 9.2% in LD dose group, 9.5%, 7.1% and 9.6% in MD dose group and 7.0%, 4.0% and 12.2% in HD dose group. All samples were homogenous, as COV was below or equal to the acceptance criterion of 20%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of a previous dose range finding study (BSL project no. 143123,) and in consultation with the sponsor doses of 100, 300 and 1000 mg/kg bw/day were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first administration and at least once a week thereafter detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were recorded as per scoring criteria.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.

FOOD CONSUMPTION
- Food consumption per housing group determined weekly and mean daily diet consumption calculated as g food/animal/day for each sex: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological parameters were examined at the end of the treatment period prior to or as part of the sacrifice of the animals.
- Anaesthetic used for blood collection: Yes (ketamine and xylazine)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cellls (Luc), prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Parameters of clinical biochemistry were examined at the end of the treatment period prior to or as part of the sacrifice of the animals.
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters examined: specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (UBG), bilirubin (BIL), blood (Ery), leukocytes (Leu)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure and once in the last week of exposure, multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / body temperature / rearing / urination / defecation
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- One day after the last administration (study day 91) all surviving animals of the treatment period were sacrificed using anesthesia (ketamine, Pharmanovo, lot no: 24863, expiry date: 10/2015 and xylazine, Serumwerk, lot no. 01213, expiry date: 10/2015) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

HISTOPATHOLOGY: Yes
- The tissues from all animals were preserved in 10% neutral buffered formalin except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol. The organs were examined histopathologically after preparation of paraffin sections paraffin sections and haematoxylin-eosin staining for the animals of groups 1 and 4 sacrificed at the end of the treatment period. These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in any organ of the high dose group. For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. Quantitative evaluation of the testes was not performed as no effect was observed in testes based on the qualitative evaluation. Any gross lesion macroscopically identified was examined microscopically in all animals.
Other examinations:
EXAMINATION OF FERTILITY PARAMETERS:
- Daily over a period of 8 days, the estrous cycle of all female animals was examined 4, 8 and 12 weeks after the first administration. At necropsy (one day after the last administration), left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser (TOX IVOS Version 13.0). Sperm morphology slides were prepared from all male animals and evaluated at BSL Bioservice. Evaluation was made in male animals of all groups sacrificed at the end of the treatment period in order to see the dose dependency of the findings for more meaningful evaluation. Therefore, sperm from left vas deferens was transferred to 0.1% bovine serum albumin solution. For staining, two drops of 1% aqueous Eosin-Y solution were mixed with six drops of the sperm-suspension. The stained sperm suspension was used to prepare smears on slides. After complete drying, the slides were dipped into 0.1% acetic acid for approximately 30 seconds to intensify the coloring.

ORGAN WEIGHT:
- The wet weight of the organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Relative organ weights were calculated in relation to brain and body weight and presented as percentage.
- Organs weighted at necropsy: liver, kidneys, adrenals, testes, epididymides, prostate (seminal vesicles and coagulating glands), ovaries, uterus with cervix, thymus, thyroid (parathyroid glands), spleen, brain, pituitary gland, heart
Statistics:
A statistical assessment of the results determined for body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry, absolute and relative organ weights and sperm analysis was performed for each gender by comparing the values of the dosed groups with those of the control group using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred in the control or any of the dose groups during the treatment period of this study and all animals were sacrificed on day 91. No test item related clinical signs were observed in any male and female animal during the entire study period. During the treatment period, slight to severe salivation was noted in a dose-dependent pattern occasionally in some males and females of the HD group and in a few of the LD and MD group. Furthermore, moving the bedding was observed transitorily in few males of the MD and HD group and few females of the LD, MD and HD group. As the symptoms of moving the bedding and salivation were noted immediately after administration and just for a short period, these signs were considered to be a local effect of the test item due to possible regurgitation of the test item rather than a systemic effect. Low incidences of slight clinical signs like alopecia, crust on various body parts, diarrhoea, reduced spontaneous activity, slight kyphosis, apathy, slight dehydration and piloerection were noted in isolated males and/or females of the dose groups and/or the control group during the treatment period. As these slight clinical signs were mostly transient and seen irrespective of the groups in isolated animals, they were considered to be incidental. In female animal no. 70 of the MD and 74 of the HD group, abnormal breathing was noted on few treatment days. No toxicological relevance is considered for this finding as this was observed in single animals of the groups and there was no effect on overall health, body weight and food consumption of these animals. During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

BODY WEIGHT AND WEIGHT GAIN
In males, mean body weight increased with the progress of the study in all groups, and mean body weight gain did not differ significantly from that in the control group, which was also correlated with the food consumption. Body weight gain was highest at the beginning of the study period and constantly decreased with time. This is considered to be a normal development as body weight gain is usually highest at young age and then constantly decreases, which also correlates with food consumption. In females, mean body weight increased with the progress of the study in all groups, and no test item related effect was observed on mean body weight and mean body weight gain in treatment groups during the entire study period. Female animals of the MD and HD group showed slightly higher mean body weights compared to the control group throughout the treatment period (between 101% and 106%), and overall body weight gain from day 1-90 was statistically significantly higher in the MD group when compared with the controls. As this difference was marginal and within the normal range of variation for this strain, it was considered to have no toxicological significance.

FOOD CONSUMPTION
No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period. In correlation to body weight gain, the food consumption in males and females was highest at the beginning of the study and then constantly decreased with the progress of the study in all groups, which is considered to be a normal development as food demand is usually highest at young age and then constantly decreases. During the whole treatment period, food consumption of the female MD and HD group was marginally higher compared to the corresponding control group, which correlates to the higher mean body weight and body weight gain of the females in these groups.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmologic findings were observed in any of the animals of this study.

HAEMATOLOGY
In males and females, no test item related effect on haematological parameters was observed in treated groups except a decrease in PLT count in LD (595.00) and MD (624.90) males when compared with the controls (649.67). However, all individual and group mean values were within the historical control data range. Due to lack of dose dependency and consistency, this finding was not considered to be toxicologically relevant. Except for PLT, all haematological parameters and blood coagulation parameters in males and females were in the normal range of variation, and no test item-related effects were observed.

CLINICAL CHEMISTRY
In male animals, a marginally but statistically significantly lower mean value for cholesterol (Chol) was noted in the male MD group (2.04) compared to the control group (2.41). As group mean and individual values for cholesterol were within the normal range of variation, and due to lack of dose dependency, this was considered as incidental. In female animals, Total Bile Acids (TBA) were marginally higher in the MD (42.15) and HD (48.20) group compared to the control group (33.93). This cannot be considered to be related to the treatment with the test item as the difference is statistically insignificant, and values were within the normal range of variation and the historical control data range. The Standard Deviation (SD) for TBA was very high in these groups, as well, and indicated a high variation in the individual values. Actually, the higher mean values of these groups were related to a few individual outliers showing considerably higher individual values for TBA compared to the other animals of the respective group. These represent isolated events and were considered to be incidental. There were no further biologically or statistically significant differences between the dose groups and the control group for all remaining parameters of clinical chemistry for male and female animals.

URINALYSIS
Slightly higher leukocyte levels were found in the urine of few male animals of all groups including the control animals. No such trend was observed in the female animals. In males and females, all other urinary parameters were in the normal range of variation and no test item-related differences between the dose groups and control group were observed.

NEUROBEHAVIOUR
No relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups. A marginal, but statistically significant lower body temperature of male HD animals before initiation of the treatment period was observed when compared to the respective controls. This effect on body temperature was considered to be incidental as it was observed before initiation of the treatment.

ORGAN WEIGHTS
In males, at the end of the treatment period, absolute and relative (to brain weight and body weight) thymus weights were slightly higher in the MD and HD group than in the respective controls without achieving statistical significance. In females, absolute and relative (to brain weight and body weight) spleen weights were higher in all treatment groups although statistical significance was only achieved for absolute spleen weight in the HD group and relative (to brain weight) spleen weights in the LD and HD group when compared with the controls. The absolute and relative (to brain weight and body weight) thymus weights in females were also higher in the HD group without achieving statistical significance when compared with the controls. In the light of absence of histopathological findings in the spleen and thymus, and as the increase was minimal, it was not considered to be adverse. All values for the remaining male and female absolute and relative organ weights from the dose groups were comparable with the control group.

GROSS PATHOLOGY
Predominant macroscopic findings observed in male animals sacrificed at the end of the treatment period were dilated renal pelvis of the kidneys (2/10 in control, 1/10 in LD, MD and HD group), swollen lung filled with ichor (1/10 in MD group) and big size thymus (1/10 in MD group). In females, few specific gross pathological changes were recorded at necropsy at the end of the treatment period. Fluid distension of uterus was noted in several females at the end of the treatment period (3/10 in C, 3/10 in LD, 2/10 in MD and 3/10 in HD group). Other gross pathological findings recorded were milky opaque eyes (1/10 in LD group), ichor around the heart (1/10 in MD group), lung swollen and filled with ichor (1/10 in MD and HD group), swollen lymphnodes of the lung (1/10 in MD and HD group) and discoloured yellowish/brownish lymph nodes of the lung (1/10 in HD group). The distributions among the groups were considered to be incidental, reflecting the usual individual variability. In addition, these gross pathological findings were spontaneous in nature and as such not considered to be a systemic effect due to test item administration. Histopathologically, these gross lesions could not be related to treatment with the test item. However, there were gross lesions in the lungs and hearts of two animals described as swollen lung filled with ichor including swollen lung lymph nodes, that correlate to abscess formation affecting also the pericardium.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathologically, the test item produced no indicator for toxicity in any of the organs and tissues examined including the reproductive system and the testes which were stained by PAS where sperm staging was performed. In the latter case, there was no finding at all recorded in the testes. There were no gross lesions or histological alterations that could be attributed to treatment with the test item. Noteworthy, gross lesions were recorded in the lungs and hearts of two animals consisting of swollen lung filled with ichor, swollen lung lymph nodes and additionally an effect on the pericardium (ichor around heart, hardening) in one case. The histological correlate consisted of abscess formation in the lungs with an inflammatory lesion extending to the heart pericardium. Such lesions are not common with other vehicles, however, in studies performed with corn oil they may be observed. The reason is deemed to be an accidental aspiration that causes focal inflammatory changes including abscesses due to the irritative effect of corn oil. Such effects however are not induced by the test item but are of accidental nature and cannot be used for the judgment of the NOAEL or NOEL. All findings, except the abscess formation in lungs with the extending inflammation to the pericardium, are within the range of spontaneous background alterations that may be recorded in Wistar rats at these ages.

FERTILITY PARAMETER
13-Docosenamide, (Z)- had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment of this study. The statistical analysis showed no statistically significant changes between the control group and any of the dose groups in the testicular number of sperms/g testis. However, a statistically significantly lower percentage of motile, rapidly moving epididymal sperms and a significantly higher static count were observed in the LD group when compared with the controls. These significant values in the LD group were attributed to low sperm motility and high static count in animal number 12 and 14. Due to lack of dose dependency and consistency, this finding was considered to be of no toxicological relevance. Evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item, and percentage of normal and abnormal sperms in treatment groups were comparable with the controls. 13-Docosenamide, (Z)- had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed occasionally, mainly as irregularly long cycles, in few animals of all treatment groups including control during the last week of the treatment. As this effect was also observed in control animals, it was considered as incidental and not related to the treatment with the test item.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no relevant treatment related effects were observed
Critical effects observed:
not specified

Table 1: Functional Observation Battery - Males - Summary

 

Group

 

 

Body Temperature (°C)

Rearing Supported (Count)

Rearing not Supported (Count)

Urination (Count)

Defecation (Count)

Before Treatment

Last Week of Treatment

Before Treatment

Last Week of Treatment

Before Treatment

Last Week of Treatment

Before Treatment

Last Week of Treatment

Before Treatment

Last Week of Treatment

 

C

Mean

38.45

36.61

5.0

2.8

0.4

0.1

0.3

0.1

1.7

0.0

SD

0.47

0.85

3.7

2.7

1.0

0.3

0.5

0.3

2.2

0.0

N

10

10

10

10

10

10

10

10

10

10

 

LD

Mean

38.07

36.97

4.3

2.7

0.2

0.3

0.2

0.0

1.5

0.0

SD

0.68

0.74

2.5

2.2

0.4

0.9

0.4

0.0

2.0

0.0

N

10

10

10

10

10

10

10

10

10

10

 

MD

Mean

37.96

37.12

3.2

3.9

0.3

0.0

0.2

0.1

1.1

0.0

SD

0.56

0.57

2.3

3.2

0.7

0.0

0.4

0.3

1.7

0.0

N

10

10

10

10

10

10

10

10

10

10

 

HD

Mean

37.59**

36.61

3.3

2.6

0.0

0.0

0.2

0.0

0.3

0.0

SD

0.64

0.46

1.6

2.2

0.0

0.0

0.4

0.0

0.7

0.0

N

10

10

10

10

10

10

10

10

10

10

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

N: Number of males

Table 2: Mean Body Weight Gain (g/day) - Females

 

Group

 

 

Day

1-8

Day

8-15

Day

15-22

Day

22-29

Day

29-36

Day

36-43

Day

43-50

Day

50-57

Day

57-64

Day

64-71

Day

71-78

Day

78-85

Day

85-90

Day

1-90

 

C

Mean

20.90

7.90

12.30

6.40

5.10

4.10

1.60

3.00

2.60

3.70

6.00

-1.60

5.40

77.40

SD

5.88

6.84

3.86

4.70

6.15

5.11

3.75

3.02

3.41

3.65

5.66

2.91

1.65

9.98

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

LD

Mean

18.30

9.20

10.60

5.10

6.50

3.80

5.70

1.70

3.30

3.60

2.10

-0.10

7.20

77.00

SD

3.47

5.09

2.72

4.23

4.72

3.58

3.20

1.95

2.67

3.31

3.03

1.45

2.35

7.69

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

MD

Mean

19.00

14.00

9.70

10.30

5.90

4.30

5.50

4.20

1.30

2.80

3.80

2.20

6.10

89.10*

SD

4.40

4.37

2.98

2.95

4.86

4.79

4.22

4.08

3.20

5.27

5.83

3.33

1.79

10.26

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

HD

Mean

19.80

11.40

12.90

5.50

5.40

4.70

3.10

1.10

3.70

2.70

3.90

0.60

8.10

82.90

SD

3.05

4.70

4.04

5.91

7.38

6.00

3.03

3.60

6.00

2.87

4.70

3.31

2.88

12.86

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

N: Number of females

Table 3: Mean Clinical Biochemistry - Males

 

Group

 

 

ASAT

ALAT

AP

Crea

TP

Alb

Urea

TBIL

TBA

Chol

Gluc

Na

K

Units

U/L

U/L

U/L

μmol/L

g/L

g/L

mmol/L

μmol/L

μmol/L

mmol/L

mmol/L

mmol/L

mmol/L

 

C

Mean

109.46

42.30

152.72

35.40

59.05

32.75

4.76

2.16

37.62

2.41

10.83

141.00

4.92

SD

28.64

10.34

42.63

16.38

1.51

1.15

0.82

0.36

25.15

0.24

1.76

1.25

0.85

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

LD

Mean

95.86

39.57

145.70

42.30

59.29

33.17

5.43

2.05

34.03

2.29

11.05

140.70

5.68

SD

28.59

10.30

49.63

30.17

1.85

0.95

1.60

0.40

17.47

0.35

5.91

1.06

1.87

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

MD

Mean

104.09

43.35

189.63

33.60

59.61

32.97

5.04

2.15

24.91

2.04*

10.40

140.90

4.98

SD

28.54

15.47

61.48

13.37

1.72

1.48

0.83

0.41

15.14

0.14

2.61

1.29

0.95

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

HD

Mean

105.35

45.69

150.61

32.60

60.74

33.59

6.11

2.09

32.49

2.40

10.06

139.90

5.03

SD

34.37

21.52

57.03

3.95

2.01

0.88

1.09

0.46

12.97

0.29

3.29

1.60

1.14

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

N: Number of males

Table 4: Mean Sperm Motility - Males

Group

 

Motile Count [%]

Static Count [%]

Rapid [%]

 

C

Mean

82.25

17.75

60.90

SD

5.19

5.19

6.18

N

10

10

10

 

LD

Mean

73.45*

26.55*

49.50*

SD

10.31

10.31

13.48

N

10

10

10

 

MD

Mean

81.35

18.65

60.90

SD

4.74

4.74

6.65

N

10

10

10

 

HD

Mean

83.00

17.00

61.25

SD

6.82

6.82

11.31

N

10

10

10

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001

C: Control

LD: Low dose

MD: Medium dose

HD: High dose

N: Number of males

Conclusions:
On the basis of the present results, the 90-Day Repeated Dose Oral Toxicity study with 13-Docosenamide, (Z)- in male and female Wistar rats, with dose levels of 100, 300, and 1000 mg/kg bw/day, revealed no major toxicity or mortality. No effects of 13-Docosenamide, (Z)- were found at the dose level of 1000 mg/kg bw/day. Therefore, the NOAEL of 13-Docosenamide, (Z)- in this study is considered to be 1000 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was carried out in accordance with an appropriate OECD test guideline and in compliance with GLP with additional focus on assessment of fertility parameters and effects on reproductive organs.

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

Repeated dose toxicity: oral

Three studies addressing the repeated dose toxicity of CAS 112-84-5 via the oral route are available.

A key subchronic repeated dose toxicity study (90-days) with CAS 112-84-5 was performed according to OECD TG 408 and in compliance with GLP (Takawale, 2015). In the available study the test substance was investigated for toxic effects after repeated administration via the oral route with a particular focus on fertility parameters and reproductive organs. Groups of 10 Wistar rats (Crl:WI(Han)) per sex per dose were administered doses of 100, 300 and 1000 mg/kg bw/day (nominal dose) via oral gavage. Animals treated with the vehicle (corn oil) served as controls. Treatment was carried out once daily (7 days per week) for 90 consecutive days. Animals were observed for mortalities and general clinical observations twice daily (except weekends). Detailed clinical observations were performed once before the first administration and at least once a week thereafter. Body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period. Ophthalmological examinations using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period. Haematology and clinical chemistry parameters as well as urinalysis were examined at the end of the treatment period prior to or as part of the sacrifice of the animals. Gross pathology and histopathology were performed one day after the last administration when all surviving animals were sacrificed. Examination of fertility parameters was performed daily over a period of 8 days. The estrous cycle of all female animals was examined 4, 8 and 12 weeks after the first administration. At necropsy the left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count were evaluated in all male animals. Moreover, the wet weight of the organs of all sacrificed animals was recorded as soon as possible. No mortality occurred in the control or any of the dose groups during the treatment period of this study. No test item related clinical signs were observed in any male and female animal during the entire study period. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. In males, mean body weight increased with the progress of the study in all groups and mean body weight gain did not differ significantly from that in the control group which was also correlated with the food consumption. In females, mean body weight increased with the progress of the study in all groups and no test item related effect was observed on body weight and body weight gain in treatment groups during the entire study period. No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period. No ophthalmologic findings were observed in any of the animals of this study. In males and females, no test item related effect on haematological and blood coagulation parameters was observed in treated groups except decrease in PLT count in LD and MD males when compared with the controls. However, all individual and group mean values were within the historical control data range. Due to lack of dose dependency and consistency, this finding was not considered to be toxicologically relevant and was considered to have no biological relevance. In male animals, a marginally but statistically significantly lower value for cholesterol (Chol) was noted in the male MD group compared to the control group. As group mean and individual values for cholesterol were within the normal range of variation and due to lack of dose dependency, this is considered as incidental. In female animals, total bile acids (TBA) were marginally higher in the MD and HD group compared to the control group. This cannot be considered related to the treatment with the test item as the difference is statistically insignificant and values were within the normal range of variation and the historical control data range. Slightly higher leukocyte levels were found in the urine of a few male animals of all groups including the control animals. In males and females, all other urinary parameters were in the normal range of variation and no test item-related differences between the dose groups and control group were observed. No relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups. A marginal, but statistically significant lower body temperature of male HD animals before initiation of the treatment period was observed when compared to the respective controls. This effect on body temperature was considered to be incidental as it was observed before initiation of the treatment. In males, at the end of the treatment period, absolute and relative (to brain weight and body weight) thymus weights were slightly higher in MD and HD group than in the respective controls without achieving statistical significance. In females, absolute and relative (to brain weight and body weight) spleen weights were higher in all treatment groups although statistical significance was only achieved for absolute spleen weight in the HD group and relative (to brain weight) spleen weights in the LD and the HD group when compared with the controls. The absolute and relative (to brain weight and to body weight) thymus weights in females were also higher in the LD and the HD group without achieving statistical significance when compared with the controls. In the light of absence of histopathological findings in the spleen and thymus and as this increase was marginal, it was not considered to be adverse. A few spontaneous gross pathological findings were observed in males and females and as such not considered to be a systemic effect due to test item administration. Histopathologically, these gross lesions were not related to treatment with the test item. No test item related effect on epididymal sperm motility or testicular sperm count was noted at the end of the treatment of this study. The statistical analysis showed no statistically significant changes between the control group and any of the dose groups in the testicular number of sperms/g testis. However, a statistically significantly lower percentage of motile, rapidly moving epididymal sperms and significantly higher static count was observed in the LD group when compared with the controls. These significant values in the LD group were attributed to low sperm motility and high static count in animal number 12 and 14. Due to lack of dose dependency and consistency, and in the absence of histopathological findings, this effect on sperm parameters is not considered to be of toxicological relevance. Evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item, and percentages of normal and abnormal sperms in treatment groups were comparable with the controls. The test material had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration. In summary, the 90-Day Repeated Dose Oral Toxicity study with CAS 112-84-5 in male and female Wistar rats, with dose levels of 100, 300, and 1000 mg/kg bw/day, revealed no major toxicity or mortality. No effects of the test material were found at the dose level of 1000 mg/kg bw/day. Therefore, the NOAEL is considered to be 1000 mg/kg bw/day.

A supporting subacute repeated dose toxicity study (28-days) with CAS 112-84-5 was performed similar to OECD TG 407 (Prier, 1960). Although the study was performed before establishment of GLP-regulations or technical guidelines, the protocol can be considered an ancestral version of OECD 407, and the study was well conducted. Therefore, it is considered as acceptable for assessment despite the obvious restrictions that only one dose without analytical confirmation was used in this study, and no histopathology was performed. The test material was administered daily to a group of five male Sprague-Dawley rats at a dose of 10% (w/w) for four consecutive weeks in the diet which was available ad libitum. Individual body weight and food consumption data were collected weekly. At the end of the study period the animals were subjected to a routine hematological investigation and urinalysis, sacrificed, and examined grossly for pathology. Tissue sections were prepared for possible future histological examination. The clinical testing and gross autopsy were all negative in that no abnormalities of any sort were noted. No mortality occurred during the study period. Animals fed of the test material in a semi-synthetic diet grew at a somewhat lower rate than fat-free controls but their food consumption was similarly lowered and the feed efficiency of the two groups was not significantly different. Since the caloric content of the two diets differed by approximately 12% on a calculated basis but by only about 4% on the basis of absorbed nutrients this lack of a real difference is not surprising. In summary, the 28-Day Repeated Dose Oral Toxicity study with CAS 112-84-5 in male Sprague-Dawley rats with a 10% (w/w) formulation in diet revealed no toxicity or mortality. Therefore, the NOAEL was considered to be 10000 ppm for males.

There is a third repeated dose toxicity study available which is not considered as acceptable for assessment (Molnar, 1960). The study was performed before GLP-regulations without following an appropriate OECD TG. The test material was not adequately characterized and no histopathology was performed. In this study a group of 10 male Wistar rats were administered ten 1 mL doses per animal/day (7500 mg/kg bw/day) of the test substance via oral gavage for five consecutive days. The animals were retained for additional 23 days after the last administration of the test material. No mortality occurred, neither gross pathology nor tissue damage was noted during the study period. No loss of body weight was recorded during the observation period. No NOAEL had been derived from this study, but a lethal dose greater than 7500 mg/kg bw had been derived.

 

In summary, the available data on repeated dose toxicity of CAS 112-84-5 does not reveal any substance-related signs of toxicity. Therefore, a subchronic NOAEL of 1000 mg/kg bw/day was derived from the 90-Day Repeated Dose Toxicity Study.

Waiving - Repeated dose toxicity: inhalation

According to Regulation (EC) No 1907/2006, Annex IX, 8.6.2, column 1, a subchronic toxicity study (90 day) has to be performed in one species (rodent, male and female) via the most appropriate route of administration, having regard to the likely route of human exposure. No subchronic inhalation toxicity study needs to be performed according to the criteria outlined in Regulation (EC) No 1907/2006, Annex IX, 8.6.2, column 2, since the vapour pressure of the substance is very low and the possibility of human exposure is not expected under normal conditions of handling. Furthermore, a 90-day oral toxicity is available for the substance (Z)-docos-13-enamide (CAS 112-84-5, erucamide), which will be used to cover the endpoint Repeated dose toxicity.

Waiving Repeated dose toxicity: dermal

According to Regulation (EC) No 1907/2006, Annex IX, 8.6.2, column 1, a subchronic toxicity study (90 day) has to be performed in one species (rodent, male and female) via the most appropriate route of administration, having regard to the likely route of human exposure. No subchronic dermal toxicity study needs to be performed according to the criteria outlined in Regulation (EC) No 1907/2006, Annex IX, 8.6.2, column 2, since the physicochemical properties (high log Pow and low water solubility) of the test substance do not suggest a significant absorption through the skin. In addition, the acute dermal toxicity and skin irritation studies conducted with the test substance according to Regulation (EC) No. 1907/2006, Annex XI, article 1.5 did not show systemic effects or evidence of absorption through the skin. Furthermore, a 90-day oral toxicity is available for the substance (Z)-docos-13-enamide (CAS 112-84-5, erucamide), which will be used to cover the endpoint Repeated dose toxicity.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The key study with a special focus on fertility parameters was selected for assessment

Justification for classification or non-classification

The available data on repeated dose toxicity of CAS 112-84-5 do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.