1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.11.15 - 30.11.16

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:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

EC Number:

700-453-0

Cas Number:

59802-05-0

Molecular formula:

C26H42O4

IUPAC Name:

1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

EST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation:
Males: approximately 71 days old.
Females: approximately 85 days old.
- Weight at study initiation:
Males: 341 to 410 g.
Females: 245 to 295 g.
- Housing:
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Number of animals per cage
Pre-pairing up to five animals of one sex
Pairing one male and one female
Males after mating up to five animals
Gestation one female
Lactation one female + litter

- Diet: Non-restricted (removed overnight before blood sampling for haematology and blood chemistry investigations and thyroid hormone analysis for F0 adults).
- Water: Non-restricted.
- Acclimation period:
Males: six days before commencement of treatment.
Females: 20 days before commencement of treatment.


DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet are scrutinised and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES: From: 02.11.2015 To: 04.07.2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Concentration in vehicle: Starting with the low concentration (20 mg/mL), the formulation was prepared by weighing out the approximately 50% of test item and adding a sufficient amount of vehicle to obtain a solution.The procedure was repeated for the mid and high dose (60 and 200 mg/mL).
- Amount of vehicle (if gavage): 5 mL/kg body weight.

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear (Dialy checks)
- After successful mating each pregnant female was caged (how): Male/female separation when mating evidence was detected.
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Week 1 and the last week of treatment were analyzed for achieved concentration of the test item

Duration of treatment / exposure:

Males: two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 (control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

10 animals / sex/ dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels of 100, 300 and 1000 mg/kg/day were selected by the Sponsor based on the results of a 14-day repeat dose preliminary study in the CD rat conducted at these laboratories (Huntingdon Life Sciences). In the preliminary study Bis (C9(branched)alkyl) benzene- 1,4- dicarboxylate was generally well tolerated at dose levels up to 1000 mg/kg/day. Effects were restricted to a slight effect on body weight for females at 300 and 1000 mg/kg/day and slight effects on food consumption for females receiving doses >300 mg/kg/day. Organ weights were considered not affected to any noticeable extent by the administration of Bis (C9(branched)alkyl) benzene- 1,4 dicarboxylate.

Positive control:

not applied

Examinations

Parental animals: Observations and examinations:

AGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Detailed observations were performed to establish and confirmed a pattern of signs in association with dosing according as follows:
F0 males Week 1 - daily
Week 2 onwards - once each week
F0 females Week 1 - daily
Week 2 onwards - once each week
Gestation phase - Day 0, 6, 13 and 20
Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day



BODY WEIGHT: Yes
- Time schedule for examinations:
The weight of animals was recorded as follows:
F0 males Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.
F0 females Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 6, 13 and 20 after mating.
Day 1, 4, 7, and 13 of lactation.
On the day of necropsy.


FOOD Consumption:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-5, 6-12 and 13-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity


OTHER: Haematology, clinical chemistry, thyroid hormone analysis, histopathology, organ weights (see Repeated dose toxicity oral)

Oestrous cyclicity (parental animals):

Dry smears For 15 days before pairing using cotton swabs.

Wet smears Using pipette lavage during the following phases:
• For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
• After pairing until mating.
• For four days before scheduled termination (nominally Day 11 to 14 of lactation).

Sperm parameters (parental animals):

Parameters examined in five lowest numbered F0 surviving males :

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weights, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after Week 5 when investigations completed.
- Maternal animals: All surviving animals on or after day the last offspring died

GROSS NECROPSY
- Gross necropsy consisted of [All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.]

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Histopathology: All animals killed or dying prematurely.
The five lowest surviving F0 surviving males and females with a surviving litter in Groups 1 and 4 at scheduled termination.

The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at day 13 of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations with an assessment of stomach for milk content. In addition and where possible, thyroid glands were preserved. Examined and abnormal tissues retained from one male and female from each litter; particular attention was paid to the external genitalia.
Thyroids were retained from one male and one female offspring in each litter on Day 13 of age.


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues and regions abnormalities and thyroids] were prepared for microscopic examination.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles, pre-coital interval and mating performance, gestation length and gestation index, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Reproductive indices:

Group values were calculated for males and females separately for the following:
Percentage mating (%)
Conception rate (%)
Fertility index (%)

Offspring viability indices:

The following were calculated for each litter:
Post-implantation survival index (%)
Live birth index (%)
Viability index (%)
Lactation index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two females receiving 1000 mg/kg/day showed a sudden decline in clinical condition during early lactation both showing common signs of hunched posture and piloerection and were killed for welfare reasons on Days 2/3 of lactation. Although no macroscopic or microscopic pathology findings were identified that could have been responsible or the sudden decline in the clinical condition of these females, the occurrence of two cases on the study both in the high dose group and both during the first 3 days of lactation is suspicious and it is concluded that a relationship to treatment cannot be completely ruled out.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment of females at all doses of Bis(C9(branched)alkyl)benzene-1,4-dicarboxylate was associated with a dose related reduction in mean body weight during the first week of treatment and slightly low food consumption during the first two weeks of treatment.In contrast, treatment of males at 1000 mg/kg/day only did not affect body weight gain during the first week of treatment but was associated with slightly but persistently low weight gain during Weeks 2-5 of treatment. During gestation, the body weight gain of females receiving 1000 mg/kg/day was slightly low between Days 13 and 20 of gestation. As a result of this, and the low weight gain during the first week of treatment, absolute body weights on Day 1 of lactation in all groups of females treated with Bis(C9(branched)alkyl)benzene-1,4-dicarboxylate were lower than in Controls. Body weight gain of females receiving 1000 mg/kg/day was low during Days 4-7 of lactation and food consumption was slightly low from Day 4 of lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption for females at 100, 300 or 1000 mg/kg/day was slightly lower than that of the Control animals in Weeks 1 and 2 of treatment before pairing. There was no effect on food consumption for females receiving Bis(C9(branched)alkyl)benzene-1,4-dicarboxylate at 100, 300 or 1000 mg/kg/day during gestation.
Food consumption for females receiving Bis(C9(branched)alkyl)benzene-1,4-dicarboxylate at 1000 mg/kg/day was slightly lower than that of Control animals for Days 4-6 and 7-12 of lactation.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematocrit, haemoglobin and red blood cell counts were statistically significantly low (p<0.05) for males receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 1000 mg/kg/day, when compared with the Control.
Reticulocyte counts were statistically significantly low (p<0.05) for males receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 100 and 300 mg/kg/day, and statistically significantly low (p<0.01) for males receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 1000 mg/kg/day when compared with the Control.
Red cell distribution widths were statistically significantly low (p<0.05) for males receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 100 mg/kg/day, and statistically significantly low (p<0.01) for males receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 300 and 1000 mg/kg/day when compared with the Control.
White blood cell, lymphocyte and basophil counts were statistically significantly low (p<0.05) for males receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 1000 mg/kg/day, when compared with the Control.
Platelet counts were statistically significantly high (p<0.05) for males receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 300 and 1000 mg/kg/day, when compared with the Control, although a dose response was not apparent. Platelet counts were also statistically significantly low (p<0.05) on Day 14 of lactation for females receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 300 and 1000 mg/kg/day, when compared with the Control, although a dose response was not apparent.

With the exception of the platelet counts, these findings occurred in one sex only, and were considered of uncertain significance to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Calcium, triglyceride and total protein concentrations were statistically significantly low (p<0.05) for males receiving Bis (C9(branched)alkyl) benzene-1,4-dicarboxylate at 1000 mg/kg/day, when compared with the Control.

These findings were considered of uncertain significance to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis: There was no clear effect of treatment with Bis (C9(branched)alkyl) benzene 1,4 dicarboxylate at 100, 300 or 1000 mg/kg/day on the thyroid hormone levels of adult males at termination, adult females on Day 14 of lactation, F1 offspring on Day 4 of age or F1 offspring on Day 13 of age. There was a high degree of inter-individual variability, with atypically high values seen for the following:
• 2 Females had atypically high plasma TSH on Day 14 of lactation
• 1 Male had atypically high plasma TSH at termination
• 1 Male had atypically high plasma T3 at termination
• 1 Female had atypically high plasma T4 on Day 14 of lactation

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effect observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of male and female offspring on Day 1 of age in the 1000 mg/kg/day group were slightly lower than in Controls.
Offspring body weight and body weight change for males and females receiving Bis(C9(branched)alkyl)benzene-1,4-dicarboxylate at 1000 mg/kg/day from Day 1-4 was similar to concurrent Control group animals, and lower from Days 4-7 and 7-13.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity was 300 mg/kg/day in view of a sudden decline in clinical condition necessitating humane kill of two females receiving 1000 mg/kg/day during early lactation for which a relationship to treatment could not be completely ruled out. There were no effects of treatment on mating performance, fertility or the post natal survival of the offspring. The NOAEL for reproductive/developmental toxicity was concluded to be 300 mg/kg/day due to a 25% reduction in offspring body weight gain at 1000 mg/kg/day.