Diethyl succinate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The NOEL (No Observed Effect Level) for general systemic toxicity, fertility and reproduction parameters was 1000 mg/kg bw/day for males and females.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-05 - 2018-09-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted 2016-07-29

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt lot No.: T101217268
- Manufacturing date: 25 Sep 2017
- Expiration date of the lot/batch: 25 Sep 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was selected as the most appropriate species being that preferred by the test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Germany, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P): 11 - 12 weeks
- Weight at study initiation: (P) Males: 380 - 447 g; Females: 251 - 299 g
- Fasting period before study: no
- Housing: Pre-mating: up to 5 of one sex/cage. Mating: 1 male/1 female/cage. Post-mating: males group caged; females individually caged
- Diet: commercially available rodent diet (Mucedola 4RF21) ad libitum
- Water: Municipal supply tap water ad libitum
- Acclimation period: 4 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2018-07-05 To: 2018-09-12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle at concentrations of 25 mg/mL, 75 mg/mL and 250 mg/mL.

VEHICLE
- Justification for use and choice of vehicle: The substance is not soluble in water therefore corn oil was used for preparing formulations appropriate for oral administration.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged how?: individually
- Any deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of formulations for concentration and homogeneity was performed twice during the study period.
Samples were taken from different places from each concentration for analysis of concentration and homogeneity. Similar sampling was undertaken from the vehicle control.
Measured concentrations were within the test facility acceptance criteria of 85 - 115% with CV < 10%.

Duration of treatment / exposure:

Males: 2 weeks prior to mating, during pairing, and continuing for a minimum of 28 days (32 days total)
Females: 2 weeks prior to mating, during pairing, continuing through gestation and parturition to 13 days post-partum (51 - 62 days total)

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males / 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: n/a

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily
- Cage side observations included: mortality and clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Males - weekly. Females - weekly then on gestation Days 0, 7, 14 and 20, Days 1, 4, 7 and 13 post-partum

FOOD CONSUMPTION INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: weekly intervals

Oestrous cyclicity (parental animals):

Oestrus cyclicity was monitored by vaginal smears in:
- all stock females for at least 2 weeks before allocation in order to exclude females with irregular cycle.
- females allocated to study starting from Day 1 of dosing up to a positive identification of copulation.
- vaginal smears were also be taken from all study females on termination.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/sex/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: yes
- for external and internal abnormalities to establish, where possible, cause of death

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after at least 28 days treatment
- Maternal animals: All surviving animals on Day 14 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ weights: Adrenal glands, Epididymides, Kidneys, Liver, Ovaries, Prostate gland, Seminal vesicles, Spleen, Testes, Thymus, Thyroid, Uterus.
- Tissues fixed and preserved: Abnormalities, Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), CVlitoral gland, Epididymides, Kidneys, Liver, Mammary area (males and females), Ovaries, Oviducts, Parathyroid glands, Pituitary gland, Prostate gland, Sciatic nerve, Seminal vesicles, Spleen, Testes, Thymus (where present), Thyroid gland, Uterus – cervix, Vagina.

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups killed on termination of treatment. After dehydration and embedding in paraffin wax, sections were cut at 5 micrometre thickness and stained with haematoxylin and eosin.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed at 14 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: external examination and sex confirmation by inspection of the gonads

Statistics:

For continuous variables such as body weight, food consumption, hormone determination and organ weight the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n iwas more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test.

Reproductive indices:

Copulation Index: No. of pairs with successful copulation/no. of pairs mated X 100
Fertility Index: No of pregnant females/no. of pairs with successful copulation X 100
Pre-coital interval: No. of nights paired prior to detection of mating
Pre-implantation loss: No. of corpora lutea – no. implantation sites/no. of corpora lutea X 100
Pre-natal loss: No. of visible implantation sites – Live litter size/no. of implantation sites X 100

Offspring viability indices:

Post-natal loss: Live litter size at birth – live litter size at Day 4/live litter size at birth X 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs were limited to hairloss and skin/fur staining, seen in female animals from all treated groups and controls during post coitum and post partum periods.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No changes in body weight were observed in treated males and females when compared to controls. A reduced body weight gain was noted in the males dosed at 1000mg/kg bw/day at the end of treatment period. This change was not associated with a significant reduction in body weight and was considered of no toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted. The sporadic lesions that were observed were considered to be an expression of spontaneous and/or incidental pathology seen in this species.

Spermatogenic cycle: Seminiferous tubules were evaluated with respect to their stage in the sperm atogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis: No changes were noted in the analysed thyroid hormones in parental males.

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: Lack of significant treatment related effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hairloss, pallor and small appearance were noted in control and treated pups with no dose-relation and were therefore considered unrelated to treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No treatment-related nor significant differences in mean pup loss were observed at birth and on Days 1, 4, 13 and 14 post partum.

Body weight and weight changes:

no effects observed

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

A very slight statistically significant reduction was noted in high dose male pups (1000 mg/kg bw/day). Due to its low severity, this change was considered to be of no toxicological significance.

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were observed in the weight of thyroid of treated pups, when compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Autolysed abdominal organs or no milk in the stomach were observed in pups found dead at birth and in pups which died during the lactation period.
Hair loss was observed in individual pups from all groups killed on Day 14 post partum.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis: No changes were noted in the analysed thyroid hormones in pups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No treatment-related nor significant differences in total litter size, live litter size, mean pup loss (percentage), mean pup weights (percentage) and sex ratio of pups were observed among the treated and the control dams at birth and on Days 1, 4 and 13 post partum.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lack of significant treatment related effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The NOEL (No Observed Effect Level) for general systemic toxicity, fertility and reproduction parameters was 1000 mg/kg bw/day for males and females.

Executive summary:

The toxic effects of diethyl succinate, as well as any effects item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring have been investigated in Sprague Dawley rats after repeated oral exposure. Doses of 0, 100, 300 and 1000 mg/kg bw/day were administered and males were treated for 14 days prior to pairing, during pairing with females and for a total of 32 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 51 to 62 days.

No effects on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring were noted at any of the dose levels investigated.

The NOAEL (No Observed Adverse Effect Level) for general systemic toxicity, fertility and reproduction parameters was therefore considered to be 1000 mg/kg bw/day for males and females.

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

Additional information

Experimental data are available from a reproductive toxicity screening study in the rat on the registered substance which investigated potential effects on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring.

The NOAEL (No Observed Adverse Effect Level) for general systemic toxicity, fertility and reproduction parameters was 1000 mg/kg bw/day for males and females.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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

According to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification and labelling is not indicated for reproductive toxicity as adverse effects on sexual function and fertility in adult males and females or evidence of developmental toxicity in offspring have not been observed.