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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2014 -- 24 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: approximately 5 weeks old.
- Mean body weight at study initiation: the males had a mean body weight of 135 g (range: 106 g to 161 g) and the females had a mean body weight of 141 g (range: 127 g to 155 g).
- Housing: polycarbonate cages with stainless steel lids; group housing
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 7 days before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 27 May 2014 to 24 June 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle. No correction factor was applied.
Test item dose formulations were prepared on a weekly basis. They were stored at room temperature protected from light and delivered to the study room at room temperature and protected from light.

VEHICLE
- Justification for use and choice of vehicle: test item soluble in the vehicle and corn oil are commonly used for this type of study.
- Concentration in vehicle: 0, 20, 60, and 200 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test item concentrations: the test item concentrations in the administered dose formulations in Weeks 1 and 4 were within the acceptable range of -0.8% to +3.1% when compared to the nominal values (± 10% of the nominal concentrations accepted).
No test item was observed in the control dose formulation.
Homogeneity: not assessed, dose formulation is a solution
Stability: stable for 10 days at room temperature and protected from light.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor based on the results of a 2 week preliminary toxicity study (NOAEL = 1000 mg/kg/day).
- Rationale for animal assignment: computerized stratification procedure based on body weight.
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment period.

CLINICAL SIGNS
- Time schedule: once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT:
- Time schedule: once before the beginning of the treatment period, on the first day of treatment and then at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week until the end of the study.

NEUROBEHAVIOURAL EXMANINATION
- Time schedule: once in Week 4 (Day 25).

HEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period for all animals.
Sacrifice and pathology:
ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all study animals

HISTOPATHOLOGY:
- all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- liver from the low- and mid-dose animals (groups 2 and 3) sacrificed at the end of the treatment period,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.
Other examinations:
no
Statistics:
yes

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs ascribed to the test item treatment.
There were 2 males at 300 mg/kg/day with loud breathing in Week 2 or 3. In absence of this clinical sign in females and in high-dose males, a relationship with the test item treatment was considered unlikely.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects on mean body weight and mean body weight change.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on mean food consumption.
The slightly higher mean food consumption noted in test item-treated groups compared with controls was considered to be fortuitous (not dose-related).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse effects in hematology parameters.
At 1000 mg/kg/day, males had a mean neutrophil count statistically significantly lower than controls (0.91 vs. 1.54 G/L, p<0.05). This had no impact on mean white blood cell count, was not associated with other variations in white blood cell subpopulations or with pathological findings and was thus considered to be of limited toxicological significance. These animals also showed a trend toward higher mean red cell parameters (packed cell volume: 0.54 vs. 0.51 L/L, red blood cell count: 9.17 vs. 8.32 T/L, hemoglobin concentration: 17.3 vs. 16.6 g/dL) associated with a trend towards a lower mean reticulocyte count (1.79 vs. 2.47%). These tendencies were slight or rather minimal and did not reach statistical significance from controls. Therefore, they were not considered to be toxicologically significant.
There were no toxicologically relevant effects in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse effects in blood biochemistry parameters.
In females treated at 1000 mg/kg/day, mean glucose and cholesterol levels were higher compared with controls (6.85 vs. 6.02 mmol/L and 1.80 vs. 1.45 mmol/L, respectively, p<0.05). As these differences were slight and not observed in the male group, they were considered to be of minimal toxicological significance.
The other variations observed in blood biochemistry were not ascribed to the test item treatment as they were not dose-related and/or had rather high standard deviations.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects on qualitative urine parameters.
In males treated at 1000 mg/kg/day, mean urine pH was lower than in controls (6.0 vs. 6.7, p<0.05). The difference was slight and not associated with any effects on the other urine parameters. It was thus considered to be of limited toxicological relevance.
There were no relevant effects in females.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Slightly increased absolute and relative-to-body liver weights were seen in females treated at 1000 mg/kg/day (respectively, +20 and +14%; p<0.01). This difference may correlate to the hepatocellular vacuolation seen microscopically.
In females treated at 100 or 300 mg/kg/day, the minimal increases in relative-to-body liver weights (+7% and +8%; p<0.05) were considered to be also probably related to test item administration, in spite of the low magnitude of these changes.
The other organ weight differences were considered to be unrelated to test item administration in view of their low magnitude, of the absence of dose-relationship and/or the absence of microscopic correlates, including the increased absolute and relative-to-body thymus weights in all test item-treated females.
The higher uterus weights in all test item-treated females were considered to be related to estrous cycle.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross changes.
A brown discoloration of pericardium with adhesion to heart was seen in one male treated at 1000 mg/kg/day. This correlated with moderate inflammation and was considered unrelated to test item administration because of the magnitude of the changes and its isolated occurrence.
There was an enlargement of the heart in one male treated at 1000 mg/kg/day that correlated to microscopic slight dilation of heart cavities. In view of the low occurrence of this finding, it was considered to be probably unrelated to test item administration.
The other few macroscopic findings were seen with similar incidence in both control and test item-treated groups and/or correlated with spontaneous findings commonly seen in the rats of these strain and age kept under laboratory condition, including white discoloration of liver, cyst in forestomach or red mandibular lymph nodes.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects at the Functional Observation Battery including motor activity.
There was a trend toward a slight increase in mean landing foot splay in females treated at 1000 mg/kg/day (77 mm vs. 59 in controls). However, this parameter has generally a high inter-individual variability. The standard deviation in the high-dose group was rather high and the trend was not confirmed by any clinical signs. A relationship with the test item treatment was considered to be unlikely.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In liver, there was minimal to slight increased vacuolation of periportal hepatocytes (see Table 4). This lesion was seen in two out of five females at 100 mg/kg/day, in four out of five females at 300 mg/kg/day, and in all females and in two out of five males treated at 1000 mg/kg/day. This finding consisted of small- to medium-sized well delimited intra-cytoplasmic vacuoles. It may correlate with slightly increased liver weights in females at 1000 mg/kg/day and to the minimal increase in cholesterol level in this group at clinical pathology. In the absence of degenerative lesions and in view of the low magnitude of this change, it was considered as a non adverse finding.
The other microscopic findings were considered to be unrelated to test item administration since there were seen with similar incidence in controls, were not dose-related and/or correlated to commonly seen background findings in the rats of these strain and age kept under laboratory condition.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Hematology data

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

White blood cells (G/L)

12.00

9.49

10.65

12.35

7.04

5.12

6.32

6.86

Neutrophils (G/L)

1.54

1.21

1.17

0.91*

0.65

0.58

0.57

0.62

Red blood cells (T/L)

Hemoglobin (g/dL)

Packed cell volume (L/L)

8.32

16.6

0.51

8.42

16.4

0.50

8.78

16.9

0.51

9.17

17.3

0.54

8.47

15.9

0.47

8.13

15.6

0.46

8.49

16.1

0.48

8.11

15.7

0.46

Reticulocytes (%)

2.47

2.74

2.31

1.79

1.90

1.61

1.97

2.24

*: p<0.05.

Table 2. Blood biochemistry data

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Glucose (mmol/L)

6.36

5.74

6.68

5.71

6.02

6.53

6.21

6.85*

Cholesterol (mmol/L)

1.68

1.69

1.57

1.74

1.45

1.32

1.72

1.80*

*: p<0.05.

Table 3. Main organ weight changes (in percentages)

 

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

100

300

1000

100

300

1000

Exam. animals / Num. of animals

5/5

5/5

5/5

5/5

5/5

5/5

Body weight

+4

0

-6

0

+2

+6

- Liver

 

 

 

 

 

 

  . absolute

-2

-2

-9

+7

+9

+20**

  . relative

-6

-3

-4

+7*

+8*

+14**

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Table 4.Test item-related microscopic finding in liver (incidence and severity)

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Examined animals / Total number of animals

5/5

5/5

5/5

5/5

5/5

5/5

5/5

5/5

Increased vacuolation; hepatocytes

 

 

 

 

 

 

 

 

. grade 1

0

0

0

2

0

2

4

2

. grade 2

0

0

0

0

0

0

0

3

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, following daily administration of the test item for 4 weeks by oral route to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day in corn oil, the No Observed Adverse Effect Level (NOAEL) was considered to be at 1000 mg/kg/day in absence of adverse effects at this dose.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item,Tricyclodecane dimethanol diacrylate, following daily oral administration (gavage) to rats for 4 weeks,according to OECD (No. 407, 3rd October 2008) and EC (No. 440/2008, B7, 30th May 2008) guidelines.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

Methods

 

Three groups of five male and five female Sprague-Dawley rats received the test item, Tricyclodecane dimethanol diacrylate, by daily oral administration for 28 days at dose-levels of 100, 300 or 1000 mg/kg/day. The test item was administered as a solution in the vehicle (corn oil) at a constant dosage-volume of 5 mL/kg/day. A control group of five males and five females received the vehicle alone under the same experimental conditions.

Test item concentrations were checked on formulations used in Weeks 1 and 4.

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. In addition, detailed clinical examinations were performed at least once weekly. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement, and hematology, blood biochemistry and urinalysis were performed on all animals.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from control- and high-dose animals sacrificed at the end of the treatment period and on all macroscopic lesions.

 

Results

The test item concentrations in the administered dose formulations analyzed in Weeks 1 and 4 were within the acceptance criteria (± 10% of the nominal values).

 

There were no unscheduled deaths, no test item-related clinical signs or effects at the Functional Observation Battery including motor activity. There were no toxicologically significant effects on mean body weight, mean body weight change or mean food consumption.

Males treated at 1000 mg/kg/day had a mean neutrophil count and a mean urine pH lower than controls (0.91vs. 1.54 G/L and 6.0 vs. 6.7, respectively, p<0.05) that were considered to be of limited toxicological significance.

In females treated at 1000 mg/kg/day, mean glucose and cholesterol levels were higher than in controls (6.85 vs. 6.02 mmol/L and 1.80 vs. 1.45 mmol/l, respectively, p<0.05) which were considered to be of minimal toxicological significance.

Increased absolute and relative-to-body liver weights were seen in females at 100, 300, and mostly at 1000 mg/kg/day. These differences correlated with microscopic non adverse increased vacuolation of periportal hepatocytes noted in these females at 100, 300 and 1000 mg/kg/day. Vacuolated hepatocytes were also observed in two males given 1000 mg/kg/day.

Conclusion

 

Under the experimental conditions of this study, following daily administration of the test item for 4 weeks by oral route to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day in corn oil, the No Observed Adverse Effect Level (NOAEL) was considered to be at 1000 mg/kg/day in absence of adverse effects at this dose.