1,4-Cyclohexanedicarboxylic acid, 1,4-diisononyl ester

Endpoint summary

Key value for chemical safety assessment

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

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD / Crl:CD(SD)

Sex:

male/female

Details on test animals or test system 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

Description of key information

One prenatal developmental toxicity study in rats according to OECD TG 414 was conducted to obtain information on possible effects of the test item DINCD on developmental toxicity.

No maternal toxicity was observed up to the highest dose level tested (1000 mg/kg/day). Moreover, no developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day). In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) were established:
Maternal NOAEL: at least 1000 mg/kg/day.
Developmental NOAEL: at least 1000 mg/kg/day.

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

Adequacy of study:

key study

Study period:

21 Dec 2020 - 05 Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

see test material information

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 201-252g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23 °C
- Humidity (%): 50-56%
- Air changes (per hr): ten or more
- Photoperiod (hrs dark / hrs light): 12h light and 12h dark

IN-LIFE DATES: From: 06 Jan 2021 To: 10 Feb 2021

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): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for concentration and homogeneity analysis. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

Duration of treatment / exposure:

From Day 6 to 20 post-coitum, inclusive

Frequency of treatment:

once daily

Duration of test:

From Day 6 to 20 post-coitum, inclusive

Dose / conc.:

0 mg/kg bw/day (actual dose received)

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:

22

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels in this study were selected based on the results of a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with Diisononyl 1,4 cyclohexanedicarboxylate by oral gavage in Sprague Dawley rats, as well as the unaudited results from treatment Days 1-36 of a 90 day study with Diisononyl 1,4 cyclohexanedicarboxylate by oral gavage in Crl:WI(Han) rats (2020-0008-DGT).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; starting on Day 6 post-coitum up to the day prior to necropsy. During the dosing period, this observation was performed post-dose.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, animals were examined on the following time points: Pretreatment Period (prior to dosing Subgroup 1 on Day 6 post-coitum), prior to dosing Subgroup 1 on Day 13 post-coitum and on the day of necropsy corresponding to subgroup.

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 21
- Organs examined: thyroid gland, uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and distribution of live and dead fetuses

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: 1ml
- Other: analysis of thyroid hormones

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups

Statistics:

Yes.
Parametric/non-parametric: one-way ANOVA F-test, Levene’s test or Kruskal-Wallis test
Incidence: Fisher's exact test

Indices:

Parental Indices and Mortality

Historical control data:

Comparison with historical control data.

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

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean serum level of total triiodothyronine (T3) was decreased (0.83x of control) at 1000 mg/kg/day but remained within the historical control range .
Mean serum level of thyroid stimulating hormone (TSH) was decreased (0.81, 0.79 and 0.75x of control, not statistically significant) at 100, 300 and 1000 mg/kg/day, but remained within the historical control range .
Mean serum level of total thyroxine (T4) was considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in thyroid weight up to 1000 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All of the recorded microscopic findings of the thyroid gland were within the range of background microscopic observations

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pre-implantation loss was increased (107% higher than control) in the 300 mg/kg/day group. This increase was considered to be the result of two animals (Nos. 53 and 56) that had a pre implantation loss of 85 and 86%, respectively. In the absence of a dose-related trend, this increase was considered unrelated to treatment with the test item.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

At scheduled necropsy, Female Nos. 5, 22 (control) and 61 (300 mg/kg/day) were non-gravid.
Therefore, the number of females with viable litters for evaluation was 20, 22, 21 and 22 in the control, 100, 300 and 1000 mg/kg/day groups, respectively.
The number of corpora lutea, implantation sites, viable and dead fetuses, early and late resorptions and post-implantation loss were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean litter sizes were 10.7, 10.5, 9.4 and 11.5 fetuses/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Anogenital distance of all rodent fetuses:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

There were no external malformations or variations noted among fetuses in any group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Among skeletal variations, mean litter incidence of supernumerary short ribs at the thoracolumbar region was increased at 1000 mg/kg/day compared to control (77.38% versus 52.36%, respectively) and exceeded the upper limit of the historical control range3. Therefore, this increase was considered related to treatment with the test item. In addition, mean litter incidences of supernumerary thoracolumbar full rib and a pelvic girdle with misaligned ilium were increased at 1000 mg/kg/day compared to control (without reaching statistical significance). As these incidences were above the historical control range , a possible relation with the test item could not be excluded.
Given their low incidences and/or in the absence of a dose-related trend, all other skeletal variations were considered not to be related to treatment with the test item.
The incidence of supernumerary short ribs at the thoracolumbar region, supernumerary thoracolumbur full rib and pelvic girdle with misaligned ilium were increased at 1000 mg/kg/day, however, as all these skeletal findings are classified as variations, these were considered not adverse.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No visceral malformations or variations were noted among fetuses that were considered to be related to treatment with the test item up to 1000 mg/kg/day.
Situs inversus was observed in one fetus (24-L2) at 100 mg/kg/day. Given the single incidence and in absence of a dose-related trend, this malformation was considered not to be related to treatment with the test item.
Given the low incidences and/or group distribution, any other visceral variations that were noted (supernumerary liver lobes and convoluted ureters) were considered not to be related to treatment with the test item.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

Description (incidence and severity):

The incidence of supernumerary short ribs at the thoracolumbar region, supernumerary thoracolumbur full rib were increased at 1000 mg/kg/day, however, as all these skeletal findings are classified as variations, these were considered not adverse.

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

No maternal toxicity was observed up to the highest dose level tested (1000 mg/kg/day). Moreover, no developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day). The incidence of supernumerary short ribs at the thoracolumbar region, supernumerary thoracolumbur full rib and pelvic girdle with misaligned ilium were increased at 1000 mg/kg/day, however, as all these skeletal findings are classified as variations, these were considered not adverse.
In conclusion, based on the results of this prenatal developmental toxicity study in time mated female Wistar Han rats the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Diisononyl 1,4-cyclohexanedicarboxylate (DINCD) were established:
Maternal NOAEL: at least 1000 mg/kg/day.
Developmental NOAEL: at least 1000 mg/kg/day.

Effect on developmental toxicity: 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 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

Within the scope of an OECD TG 422 reproductive/developmental screening study and a prenatal developmental toxicity study according to OECD TG 414, no classification for reproductive or developmental toxicity is indicated for DINCD according to the classification, labeling, and packaging (CLP) regulation (EC) No 1272/2008.