Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Bis (C9(branched)alkyl) benzene- 1,4-dicarboxylate: Combined Repeated Dose Toxicity Study and Reproductive/Developmental Toxicity Screening Study in the CD Rat by Oral Gavage Administration
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

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:
TEST 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
Details on route of administration:
Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Vehicle:
corn oil
Details on oral 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.
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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
Group 1 (control)
Dose / conc.:
100 mg/kg bw (total dose)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw (total dose)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw (total dose)
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

Observations and examinations performed and frequency:
CAGE 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.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at the termination of the study: The five lowest numbered surviving males and females per group
- Anaesthetic used for blood collection: Yes by isoflurane
- Animals fasted: Yes
- Following parameters were examined:
Hematocrit (Hct)
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)
Mean cell hemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study termination
- Animals fasted: Yes
- How many animals: The five lowest numbered surviving males and females per group
- Following parameters were examined:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)


URINALYSIS: No


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

IMMUNOLOGY: No


OTHER: Estrous Cycles, Gestation, Records Made During Littering Phase, Thyroid hormone analysis, histopathology, organ weights (see Toxicity to reproduction)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

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.
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):
not examined
Food efficiency:
effects observed, non-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.
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
Description (incidence and severity):
The following intergroup differences from Control in organ weights were apparent: higher adjusted kidney weights in males receiving 1000 mg/kg/day; higher adjusted liver weights for males receiving 100, 300 or 1000 mg/kg/day; lower absolute and adjusted thymus weights for males and females receiving 1000 mg/kg/day.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
no
Treatment related:
no
Dose response relationship:
no
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.