Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-09 to 2016-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Preliminary Dose Range Finding Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
Preliminary pre-natal dose range finding study (no guideline required)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: INTERBUSINESS S.r.l. (Via Spartaco, 2520135 Milano, Italy); Batch no. 6061
- Expiration date of the lot/batch: 2018-03-01
- Purity test date: 2016-03-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The required amount of INTERCURE®1 was suspended in the vehicle. The formulation wasprepared daily (concentrations of 2, 20 and 200 mg/mL). Concentrations were calculated andexpressed in terms of test item as supplied.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : suspended in the vehicle (corn oil)

OTHER SPECIFICS:
Identity: INTERCURE®1
Alternative name Hexamethylenediamine carbamate
CAS No.: 143-06-6
EC No.: 205-581-6

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Hsd
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl (San Pietro al Natisone (UD), Italy)
- Age at study initiation: females: 9 weeks old; males: 11 weeks
- Weight at study initiation: females: 200-225 g; males: at least 350 g
- Fasting period before study: Not specified
- Housing: Before and after mating, the animals were housed no more than 5 of one sex to a cage in clear polysulfone cages measuring 59.5 × 38 × 20 cm; nesting material was provided inside suitable bedding bags. During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5 × 26.6 × 18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy) ad libitum
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: approximately 4 weeks

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

IN-LIFE DATES: From: 2016-04-09 To: 2016-06-02

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The required amount of INTERCURE ® 1 was suspended in the vehicle. The formulation was prepared daily (concentrations of 2, 20 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE: Corn oil
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: 2, 20, and 200 mg/mL
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: Females were paired one to one in the home cage of the male and left overnight.
- M/F ratio per cage: 1:1
- Length of cohabitation: until a positive identification of mating was made.
- Further matings after two unsuccessful attempts: Not specified
- Verification of same strain and source of both sexes: Not specified
- Proof of pregnancy: sperm in the vaginal smear or by the presence of a copulation plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
From Day 6 through Day 19 post coitum.
Frequency of treatment:
All animals were dosed once a day.
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
10 mg/kg bw/day
Remarks:
Low
Dose / conc.:
100 mg/kg bw/day
Remarks:
Medium
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High (stopped after 4/5 days of administration based on unexpected severe toxicity)
No. of animals per sex per dose:
6 female rats/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels selected in consulatation with the Sponsor

- Rationale for animal assignment (if not random): On the day of allocation (Day 0 post coitum) all females were weighed and allocated to thegroups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 to a cage. The cages were identified by a label recording the study number,animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

Mortality: Animals were checked early on each working day and again in the afternoon. On weekends and Public Holidays, the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Each animal was observed daily andany clinical signs recorded from allocation until sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15 and 20post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data provided

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data provided

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20 (All animals, including those found dead, were subjected to necropsy, supervised by a pathologist)
- Organs examined: detailed post mortem examinations were conducted (including examination of the external surface and orifices). Changes were noted and the abnormalities preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands which were fixed in Davidson’s fluid). See Table ?.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not obtained from animals found dead or killed during the study)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- number, sex and weight of all live foetuses;
- number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing);
- number of intra-uterine deaths;
- gross evaluation of placentae.
Fetal examinations:
- External examinations: Yes: All viable foetuses per litter
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

All viable foetuses were euthanised and externally examined to identify:

- Major abnormalities that are rare and/or affect the survival or health of the species under investigation
- Minor abnormalities that are detected relatively frequently
- Changes that occurs within the normal population under investigation and is unlikely to adversely affect survival or health (i.e delay in growth or morphogenesis)
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the KruskalWallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Indices:
Litter data

1) Pre-implantation loss was calculated as a percentage from the formula:

Pre impl. Loss% = ((No. of corpora lutea – no. of implantations) / no. of corpora lutea) x 100

2) Post-implantation loss was calculated as a percentage from the formula:

Post impl. Loss% = ((No. of implantations – no. of live foetuses) / no. of implantations) x 100

3) Total implantation loss was calculated as a percentage from the formula:

Total impl. Loss% = ((No. of corpora lutea – no. of live foetuses) / no. of corpora lutea) x 100

4) Sex Ratio: Sex ratios of the foetuses were calculated as the percentage of males per litter.
Historical control data:
Not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were observed in animals treated at 10 and 100 mg/kg/day.

Signs of severe toxicity such as decreased activity, kyphosis, piloerection, yellow staining, pallor and dyspnoea, semiclosed eyes and ocular discharge, were observed in high dose animals. Soft faeces were seen in cages of high dose females (1000 mg/kg/day) starting from Day 8 post coitum, up until the unscheduled termination.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female of the high dose group was sacrificed for humane reasons on Day 9 post coitum, and two females of the same group were found dead on Days 10 and 11 post coitum. Necropsy of these animals showed abnormal size (distended) and abnormal contents (gas/red fluid/red soft/white mucoid) of the gastrointestinal tract, abnormal areas (red/dark/depressed) in the glandular and non glandular region of the stomach. Yellow staining of the perianal region in the humanely killed female and abnormalred colour of the thymus in one of the found dead females were also observed. Due to the unexpected high mortality observed, treatment of the remaining three females was suspended and animals sacrificed due to abnormal red colour and/or small size of the mesenteric lymph nodes, abnormal red colour of thymus, yellow staining of the urogenital region and abnormal yellow mucoid content of intestinaltract.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No relevant differences were recorded in body weight during the study in low and mid-dose groups, when compared with controls. A statistically significant decrease in body weight (-11%) was seen on Day 9 post coitum in high dose females, when compared with controls. No relevant differences were seen in body weight gain between treated (low and mid-dose) and control groups.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences in terminal body weight, gravid uterus weight and absolute weight gain were observed in treated groups, when compared to the control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities were detected at termination in females receiving 10 and 100 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS: No clinical signs were observed in animals treated at 10 and 100 mg/kg/day.

Signs of severe toxicity such as decreased activity, kyphosis, piloerection, yellow staining, pallor and dyspnoea, semiclosed eyes and ocular discharge, were observed in high dose animals. Soft faeces were seen in cages of high dose females (1000 mg/kg/day) starting from Day 8 post coitum, up until the unscheduled termination.

MORTALITY: One female of the high dose group was sacrificed for humane reasons on Day 9 post coitum, and two females of the same group were found dead on Days 10 and 11 post coitum. Necropsy of these animals showed abnormal size (distended) and abnormal contents (gas/red fluid/red soft/white mucoid) of the gastrointestinal tract, abnormal areas (red/dark/depressed) in the glandular and non glandular region of the stomach. Yellow staining of the perianal region in the humanely killed female and abnormalred colour of the thymus in one of the found dead females were also observed. Due to the unexpected high mortality observed, treatment of the remaining three females was suspended and animals sacrificed due to abnormal red colour and/or small size of the mesenteric lymph nodes, abnormal red colour of thymus, yellow staining of the urogenital region and abnormal yellow mucoid content of intestinal tract.

BODY WEIGHT AND WEIGHT CHANGES: No relevant differences were recorded in body weight during the study in low and mid-dose groups, when compared with controls. A statistically significant decrease in body weight (-11%) was seen on Day 9 post coitum in high dose females, when compared with controls. No relevant differences were seen in body weight gain between treated (low and mid-dose) and control groups.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Dead fetuses:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Remarks on result:
other: Dose range finding study

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Mortality and systemic toxicity
Description (incidence and severity):
Signs of severe toxicity such as decreased activity, kyphosis, piloerection, yellow staining,pallor and dyspnoea, semi closed eyes and ocular discharge observed in high dose animals. Additionally, a statistically significant decrease in body weight (-11%) was seen on Day 9 post coitum in high dose females, when compared with controls.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean foetal weight was not affected by treatment.

Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Litter data was not affected by treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Litter sex ratios were not affected by treatment.
External malformations:
no effects observed
Description (incidence and severity):
External examination of foetuses did not show abnormalities with the exception of one small foetus in each treated group as well as in control group.
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Litter data, mean foetal weight and sex ratios were not affected by treatment. No abnormalities were detected in foetuses of treated females with the exception of onesmall foetus in each treated group as well as in control group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Remarks on result:
other: Dose Range Finding study

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 2. Group Incidence

 

Group

1

2

3

4

Initial Group Size

6

6

6

6

Not Pregnant

0

0

0

1

Humanely Killed

0

0

0

4

Found Dead

0

0

0

2

With live Foetuses at Gestation Day 20

6

6

6

-#

#: unscheduled death

Table 3. Body Weight – Group Mean Data

Group (s)

 

Day of Phase

0!

3’’

6

9

12

15

20

1

(n)

6

6

6

6

6

6

6

Mean

249.52

268.04

273.87

285.41

299.41

319.66

402.40

SD

6.40

10.63

6.62

8.21

8.13

12.28

21.43

 

2

(n)

6

6

6

6

6

6

6

Mean

249.22

268.80

276.86

289.36

302.48

322.57

405.33

SD

8.76

8.78

11.67

11.88

14.32

22.51

41.69

 

3

(n)

6

6

6

6

6

6

6

Mean

250.33

263.46

274.19

280.83

295.17

317.94

404.89

SD

10.47

10.58

10.17

11.52

10.81

10.26

10.69

 

4

(n)

4

4

4

4

 

 

 

Mean

249.05

264.90

275.07

256.29**

 

 

 

SD

12.68

13.21

13.53

15.67

 

 

 

! = Gestation phase; " = Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05 

** = mean value of group is significantly different from control at p < 0.01 

Statistical analysis: Dunnett`s test if group variances are homogeneous; Modifiedttest if group variances are inhomogeneous ($)

Table 4. Litter Data and Sex Ratios of Females – Group Mean Data

Group (s)

 

Corpora

Lutea

Implantations

Uterine Deaths

Viable Young

%

Males

Implantation Loss

Litter

Weight

(g)

Mean Foetal Weight

(g)

Early

Late

Total

Total

Male

Female

Pre

Post

Total

1

Mean

15.0

15.0

0.7

0.0

0.7

14.3

6.7

7.7

46.6

0.0

5.0

4.9

54.4

3.8

SD

2.4

2.4

0.8

0.0

0.8

2.9

1.9

2.2

8.9

0.0

6.6

6.6

8.6

0.4

N

6

6

6

6

6

6

6

6

6

6

6

6

6

6

 

2

Mean

14.8

14.8

0.5

0.0

0.5

14.3

8.0

6.3

55.1

0.0

3.0

3.0

55.4

3.8

SD

4.4

4.4

0.8

0.0

0.8

4.2

2.8

2.0

7.1

0.0

4.8

4.8

19.4

0.6

N

6

6

6

6

6

6

6

6

6

6

6

6

6

6

 

3

Mean

14.2

14.2

0.0

0.0

0.0

14.2

6.3

7.8

45.5

0.0

0.0

0.0

52.8

3.8

SD

2.2

2.2

0.0

0.0

0.0

2.2

1.2

2.6

9.5

0.0

0.0

0.0

4.8

0.3

N

6

6

6

6

6

6

6

6

6

6

6

6

6

6

* = Statistically significantly different from control group value

 

Table 5. External Examination of Foetuses – Group Mean Data

Group

Organ

Cat

Observation (s)

No. of Foetuses

No. of Litters

Observed

Affected

%

Observed

Affected

%

1

Whole Foetus

 

No abnormalities detected

86

85

98.84

6

-

-

Whole Foetus

AN

Small

86

1

1.16

6

1

16.67

2

Whole Foetus

 

No abnormalities detected

86

85

98.84

6

-

-

Whole Foetus

AN

Small

86

1

1.16

6

1

16.67

3

Whole Foetus

 

No abnormalities detected

85

84

98.82

6

-

-

Whole Foetus

AN

Small

85

1

1.18

6

1

16.67

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in this dose range-finding study, it was concluded that the test material exhibited maternal toxicity when administered at 1000 mg/Kg/day, while it was well tolerated when administered to pregnant rats for 14 days (Days 6-19 post coitum), up to the dose level of 100 mg/Kg/day.

 
Executive summary:

In a key pre-natal developmental toxicity dose range-finding study, the test material was administered once daily via oral gavage to pregnant Sprague-Dawley rats (6/dose) at doses of 10, 100 and 1000 mg/Kg/day for a period of 14 days (Days 6-19 post coitum). The treatment of the high dose group (1000 mg/Kg/day) was stopped after 4/5 days of administration due to unexpected severe toxicity observed (one female was sacrificed for humane reasons (Day 9 post coitum) and two females were found dead (Days 10 and 11post coitum). In agreement with the Sponsor, the remaining females were sacrificed on Days 10/11post coitum. The remaining two groups of 6 mated female rats received the test material at dose levels of 10 and 100 mg/Kg/day for 14 days (Days 6-19 post coitum), while a concurrent control group received the vehicle (corn oil).

Body weight and daily clinical signs were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examinations. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities.

 

All females in control and treated groups were pregnant with live foetuses at gestation Day 20, with the exception of one high dose female. One female in the high dose group was sacrificed for humane reasons on Day 9 post coitum, and two females of the same group were found dead on Days 10 and 11 post coitum. Abnormal size (distended) and abnormal contents (gas/red fluid/red soft/white mucoid) of the gastrointestinal tract, abnormal areas (red/dark/depressed) in the glandular and non glandular region of the stomach, were observed in these females. In addition, yellow staining on the perianal region in the humanely killed female and abnormal red colour of the thymus in one of the found dead females, were also seen. In two of the three high dose females sacrificed to interrupt the treatment, abnormal red colour and/or small size of the mesenteric nodes, abnormal red colour of thymus, yellow staining of the urogenital region and abnormal yellow mucoid content of intestinal tract was observed.

 

No clinical signs were observed in animals treated at 10 and 100 mg/Kg/day. Signs of severe toxicity such as decreased activity, kyphosis, piloerection, pallor and dyspnoea, semi closed eye and ocular discharge, were observed in animals dosed at 1000 mg/Kg/day,only. Soft faeces were seen in cages of high dose females starting from Day 8 post coitum, up until the unscheduled termination.

 

No relevant differences were recorded in body weight during the study in females treated at 10 and 100 mg/Kg/day, when compared with controls. A statistically significant decrease in body weight (-11%) was seen on Day 9 post coitum in females treated at 1000 mg/Kg/day, when compared with controls. No relevant differences were seen in body weight gain between low and mid-dose groups and control. A statistically significant decrease in body weight gain was seen on Day 9 post coitum in females treated at 1000 mg/Kg/day, when compared with controls.

 

No significant differences in terminal body weight, gravid uterus weight and absolute weight gain were observed in treated groups, when compared to the control group and no abnormalities were detected at the macroscopic evaluation of females treated at 10 and100 mg/Kg/day.

 

Litter data, mean foetal weight and sex ratios were not affected by treatment. No external abnormalities were detected in the foetuses. The incidence of small foetuses was similar in treated and in control groups.

 

On the basis of the results obtained in this study, it was concluded that the test material exhibited maternal toxicity when administered at 1000 mg/Kg/day, while it was well tolerated when administered to pregnant rats for 14 days (Days 6-19 post coitum), up to the dose level of 100 mg/Kg/day.