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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
COMBINED REPEATED DOS E TOXICITY STUDY WITH THE REPRODUCTION / DEVELOPMENTAL TOXICITY SCREENING TEST
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.12.2016 to 23.11.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

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

Test animals

Species:
rat
Strain:
other: CD(R) / Crl:CD(SD
Sex:
male/female
Details on test animals and environmental conditions:
Test species / Strain / Stock Rat / CD(R) / Crl:CD(SD)
Breeder: Charles River Laboratories, Research Models and Services, Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
Body weight (at 1st administration on TD15): Males: 398.1 g - 476.9 g; Females: 233.9 g - 291.0 g
Age (at 1st administration on TD15): Males and Females: 80 days
Selection of species: The rat is a commonly used rodent species for such studies.

Number and sex of animals:
Pre-exposure period (TD 1 to TD 14): 65 female animals
A sufficient number of animals in order to grant at least 40 females with a normal oestrus cycle
for the main study and 10 for the toxicity study.
Main study (start on TD 15): 80 animals (40 males and 40 females)
A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the FO generation.
Toxicity study with recovery period (start on TD15) 20 animals (10 male and 10 female)
Adaptation period: 5 days

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Route of administration: Oral, by gavage.
Frequency of administration: Once daily.
Administration volume 2 mL/kg b.w./day
Vehicle: Corn oil
Selection of route of administration: According to international guidelines.
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Main study animals
Males: 2 weeks prior to mating (from test day 15 until test day 29), during
the mating period (from test day 30 until test day 41 at maximum)
and during the post-mating period until and including test day 48
(one day before sacrifice on test day 49) (i.e. 34 treatment days).
Females: (with litter) 2 weeks prior to mating (from test day 15 until test day 29), during
the mating period (from test day 30 until test day 41 at maximum)
and during the lactation period until and including test days 64 to 75
(1 to 3 days before sacrifice on test days 65 to 76) (i.e. 50 to 61 treatment days).

Toxicity study animals (with recovery period)
Males: From test day 15 until and including test day 48 (i.e. 34 treatment days).
Followed by a recovery period from test day 49 until test day 66.
Females: From test day 15 until and including test day 65 (i.e. 51 treatment days).
Followed by a recovery period from test day 66 until test day 80.
Frequency of treatment:
Once daily (from test day 15 (first day of administration) until one day before sacrifice of the last female animal of the main study)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control (vehicle))
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
Pre-exposure period: 65 females
A sufficient number in order to obtain at least 40
females with normal oestrus cycles for the main
study and 10 females for the toxicity study.

Main study: 80 animals (40 males and 40 females)
A sufficient number in order to grant at least 8
females per group for evaluation of F0 generation.

Toxicity study with recovery period
20 animals (10 males and 10 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor based on available toxicological data and a 14-day dose range finding study.
In the 14-day dose range finding study, Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) was administered orally to male and female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day for 2 weeks.
No premature deaths, no abnormalities in behaviour, the external appearance or the condition of the faeces and no test item-related influence on body weight or food consumption were noted for the animals of the treatment groups (100, 300 or 1000 mg/kg b.w./day).The macroscopic inspection of the inner organs and tissues revealed no pathological changes. Furthermore, no test item-related influence on the organ weights was noted for the animals of the treatment groups (100, 300 or 1000 mg/kg b.w./day).
Based on the data obtained in this dose range finding study, dose levels of 100, 300 and 1000 mg Diisononyl 1,4-cyclohexanedicarboxylate (DINCD)/kg b.w./day were selected for the present study.

Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all adult animals.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum) and on day 4 and 13 post-partum. Body weights were recorded individually for each adult animal.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Main study animals
5 male and 5 female animals randomly selected from each group: At the end of the premating period on test day 29 (male and female animals)

Toxicity study animals (recovery)
Male animals
(all per group = 5 animals): On test day 66 (day of sacrifice).
Female animals
(all per group = 5 animals): On test day 80 (day of sacrifice).

- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes
- Parameters:
Haemoglobin content (HGB)
Erythrocytes (RBC)
Leucocytes (WBC)
Differential blood count (relative and absolute)
Reticulocytes (Reti)
Erythrocytes Platelets (PLT)
Haematocrit value (HCT)
Mean corpuscular volume
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Thromboplastin time (TPT)
Activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see under hematology
- Animals fasted: Yes
- How many animals: see under hematology
- Parameters checked were examined:

Albumin
Globulin
Albumin/Globulin ratio
Bile acids
Bilirubin (total)
Cholesterol (total)
Creatinine
Glucose
Protein (total)
Blood urea (BUN)

Calcium
Chloride
Potassium
Sodium
Sodium/Potassium ratio
BUN/Creatinine ratio
Lactate dehydrogenase (LDH)
Alanine
aminotransferase
(ALAT)
Alkaline
phosphatase
(aP)
Aspartate
aminotransferase
(ASAT)

URINALYSIS: Yes
- Time schedule for collection of urine:
Main study animals
5 male animals randomly selected from each group. At the end of the premating period on test day 29.

Toxicity study animals
All male animals On test day 29; At the end of the recovery period (test day 66)

- Metabolism cages used for collection of urine: Yes, The urine was collected for 16 hours in a URIMAX funnel cage.
- Animals fasted: Yes
- Parameters checked were examined:
Volume
pH
Specific gravity
Protein
Glucose
Bilirubin
Urobilinogen
Ketones
Haemoglobin (Hb) (approx. values)
Nitrite
Epithelial cells
Leucocytes
Erythrocytes
Organisms
Further constituents (i.e. sperm, casts)
Crystalluria

NEUROBEHAVIOURAL EXAMINATION: Yes
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad ), as well as the assessment of grip strength (Meyer et. al. ) and motor activity assessment were conducted as described on the following pages in five males and five females randomly selected from each study group.
The screening was conducted two hours after dosing and before any blood sampling for laboratory examinations:
Main study animals (shortly before scheduled sacrifice)
5 parental male animals per group
(randomly selected) On test day 48.

5 parental female animals per group
(randomly selected) Between test days 64 and 73.

Toxicity study animals with recovery period (at the end of the recovery period)
Male animals of the toxicity study
(all per group = 5 animals) On test day 63.

Female animals of the toxicity study
(all per group = 5 animals) On test day 78.


- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
DETERMINATION OF THYROID HORMONS:
1.) Thyroxine (T4):
-Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day, as scheduled below:
Pups(at least 2 surplus pups per litter, all litters): On PND 4 and 13
All evaluated dams/ all adult males: At scheduled sacrifice
-measuring instrument: T4 ELISA (Total Thyroxine (T4) ELISA, IBL INTERNATIONAL cat. no. RE55261; batch nos. 304K096 and 304K096-2; Instrument: Tecan Sunrise)

2.) Thyroid-stimulating hormone (TSH) determination:
-The TSH level were determined in the serum samples (double determination) obtained from pups on PND 13 -
-measuring instrument: The Thyroid-stimulating hormone (TSH (rat)) ELISA, IBL INTERNATIONAL cat. no. RE45021; batch nos. V028 and V031; Instrument: Tecan Sunrise)

REPRODUCTIVE PERFORMANCE (see 7.8.2)
Sacrifice and pathology:
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Main study animals
Males On test day 49 (including 34 treatment days)
Pups On PND 13
Females with litter Between PND 14 and 16
Toxicity study
Males On test day 66 (after a recovery period of 17 test days)
Females On test day 80 (after a recovery period of 14 test days)


GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increased cholesterol concentrations were noted for the male and the female animals in all treatment groups, more pronounced for the female animals than for the male animals. However, as all cholesterol concentrations of the individual female animals were within the LPT background range, this observation was considered as spontaneous and not as test item-related.

Further statistically significant changes that were not considered as test item-related or of toxicological relevance was Alkaline phosphatease (decreased in group 3 females on test day 29).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
However, on test day 29 a slight reduction in the pH value was noted for the main study animals of the intermediate and the high dose group and the high dosed animals of the toxicity study, statistically significant for the main study animals at p ≤ 0.01. As fasting results in a reduction of the pH value of the urine in general, the slightly more pronounced reductions of the pH value that were noted at the high dose group and / or the intermediate dose group in comparison to the control group were considered as spontaneous and in the range of normal variability. This was demonstrated by the pH value that was noted for the animals of the control group at the end of the recovery period on test day 66. This pH value was even slightly below the pH value that was noted for the high dosed animals on test day 29 (pH 6.04 in comparison to 6.10).
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
For the female animals slightly reduced absolute and relative heart weights were noted at the high dose level, statistically significant for the absolute heart weight.
As the reduction of the heart weight at the high dose level in comparison to the control group was only slight and all values of the absolute female heart weight at the high dose level were within the LPT background range, this change was considered as spontaneous and not as test item-related.
Furthermore, no differences in the absolute and the relative heart weight were noted between the female animals of the control and the high dose group of the toxicity study at the end of the recovery period.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following changes were considered as spontaneous and not test item-related:
Male rats
- A cyst in the right kidney of animal no. 51 of the intermediate dose group.
- A reddened discoloured thymus of animal no. 78 of the high dose group.

Female rats
- A small adrenal gland from animal no. 61 of the intermediate dose group.
- The non-pregnant animal no. 88 of the high dose group revealed a partly pale liver. Furthermore, the cervix and the left uterus horn were thickened.
- An irregular shaped spleen with a rough surface was noted for animal no. 94 of the high dose group.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone (T4) determination:
A slight decrease of 15.6% was detected in thyroxine serum levels of the dams after treatment with 1000 mg test item/kg b.w./day. The individual values of the dams showed certain variability. In addition, no dose-response relationship was observed. For this reason, the decrease is considered as spontaneous and not test item-related.
No test item-related differences were noted between the male and female animals of the control group and the male animals of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest tested dose
Dose descriptor:
NOAEL
Remarks:
reproductive parameter of the parental females
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest tested dose

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, the OECD guideline 422 with the test item Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) revealed that the NOAEL for systemic and reproductive toxicity is above 1000 mg test item/kg b.w./day, p.o..
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity,

reproduction and/or development according to OECD guideline 422. The test item Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) was

administered orally to rats at dose levels of 100, 300 or 1000 mg test item/kg b.w./day.

In order to investigate the reversibility of potential signs of general toxicity during

the main study, a toxicity study with a recovery period was additionally performed.

General toxicity - Main study and toxicity study

No premature deaths were noted.

No changes were noted in behaviour, the external appearence or the condition of the faeces.

The neurological screening, the body weight, the food consumption and the

examined haematological, biochemical and urinary parameters revealed no test

item-related differences between the control group and the treatment group.

The final examinations revealed no test item-related findings during the

macroscopic inspection at necropsy, the examination of the organ weights, the

examination of the T4 level and the microscopic examination.

Reproductive toxicity

Parantel - Generation

No test item-related influence was noted on the fertility and the gestation indices,

the pre-coital time and the gestation length.

Pups

No test item-related influence on prenatal development (post-implantation loss,

number of pups born, number of stillbirths, birth and live birth indices) were noted

at any of the tested dose levels.

No test item-related influence onpostnatal development(pup body weight, viability index, the sexual development (ano-genital distance, male nipples counting), gross abnormalities, T4serum levels of pups on lactation days 4 and 13 and TSH serum levels of pups on lactation day 13) was noted at any of the tested dose levels.

The following no-observed-adverse-effect levels (NOAEL) were established:

General toxicity

NOAEL for systemic toxicity above 1000 mg test item/kg b.w./day, p.o.

Reproductive toxicity

a) for reproductive parameters of the parental females

NOAEL above 1000 mg test item/kg b.w./day, p.o.

b) for pre- and postnatal development

- b1) adverse effects on prenatal development (conceptus to birth)

NOAEL above 1000 mg test item/kg b.w./day, p.o.

- b2) adverse effects on postnatal development (pup)

NOAEL 1000 mg test item/kg b.w./day, p.o.