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Effects on fertility

Description of key information

One repeated dose toxicity study according to OECD guideline 422 was conducted to obtain information on possible effects of the test item DINCD on general toxicity, reproduction and/or development .

No influence was noted on the fertility and the reproductive parameters of the parental generation (F0-Generation) with respect to fertility, pre-coital time, gestation length, gestation index, number of stillbirths, birth and live birth index and the preand post-implantation loss for any of the examined dose levels.

The NOAEL for adverse effects on fertility and reproduction parameters is above the highest administered dose 1000 mg test item/kg b.w./day, p.o..

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CD / 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
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount of vehicle (if gavage): 2 mL/kg b.w./day
Details on mating procedure:
- M/F ratio per cage: monogamous
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): single-housing on the other side of the room with each dose group separated by an empty row.
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
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)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
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
Parental animals: Observations and examinations:
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)

Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight,
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 and all male animals of the toxicity study following H-E and PAS staining.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
After counting on PND 4, the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups following a randomization scheme.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts, postnatal mortality, presence of gross anomalies, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups, thyroid hormone determination

GROSS EXAMINATION OF DEAD PUPS:
no

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weight of the following organs was determined before fixation (where applicable):
Adrenal gland (2) Spleen
Brain Testicle (2)
Epididymis (2) Thyroid
Heart Thymus
Kidney (2) As a whole: prostate, seminal vesicles
Liver with coagulating glands
Ovary (2) Uterus including cervix
Postmortem examinations (offspring):
SACRIFICE
At day 13 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Only for thyroid
Reproductive indices:
Female fertility Index [%]
Gestation Index [%]
Birth Index [%]
Live Birth Index [%]
Viability Index [%] (pre-cull)
Viability Index [%] post cull)
Post-implantation loss [%]
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
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).
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: higest tested dose
Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Analysis of T4 level -Pups (Postnatal day 13):
Decreased T4 concentrations were noted for the pups of the high dose group (1000 mg test item/kg b.w./day) on lactation day 13 in comparison to the control group, statistically significant at p ≤ 0.01). Under consideration of the high variability in the T4 serum level of the dams (see Po) and on lactation day 4, this decrease is considered as not test item-related and in the range of normal variability.
The determination of the T4 concentration in the serum from pups from lactation days 4 and 13 revealed no test item-related differences between the control group and the low and the intermediate dose group (100, 300 or 1000 mg test item/kg b.w./day).
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
for prenatal and postnatal development
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest tested dose
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Remarks:
Prenatal developmental Toxicity Study in rats by oral administration according to OECD guideline 414
Type of information:
experimental study planned
Study period:
To be determined by ECHA
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
A Prenatal Developmental Toxicity study for the first species is conducted as a standard information requirement as laid down in Annex IX, Section 8.7.2. of the REACH Regulation. The endpoint "Developmental toxicity/teratogenicity" will again evaluated if the complete data set of the OECD TG 414 (first species in rat) is present.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Diisononyl 1,4-cyclohexanedicarboxylate (DINCD)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: A 28-day oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test is available, which was conducted according to OECD TG 422, and in compliance with GLP.
- Available non-GLP studies: none available for toxicity to reproduction endpoint
- Historical human data: no data
- (Q)SAR: no data
- In vitro methods: no validated alternative in vitro methods are available
- Weight of evidence: insufficient data existing
- Grouping and read-across: no read-across data available


CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- There are no column 2 adaptations for repeated dose toxicity

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: a Prenatal Developmental Toxicity study in rodents according to OECD TG 414 will be conducted with Diisononyl 1,4-cyclohexanedicarboxylate (DINCD).
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Species:
rat
Route of administration:
oral: gavage
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

No classification for reproductive toxicity is indicated according to the classification, labeling, and packaging (CLP) regulation (EC) No 1272/2008.