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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 24 March 2017 and 06 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction mass of bis(2-ethylhexyl) terephthalate, butyl 2-ethylhexyl terephthalate, and dibutyl terephthalate
EC Number:
946-149-3
Cas Number:
1571954-81-8
IUPAC Name:
Reaction mass of bis(2-ethylhexyl) terephthalate, butyl 2-ethylhexyl terephthalate, and dibutyl terephthalate
Test material form:
liquid
Details on test material:
Identification: GL500
Test item (alternative names): LGflex GL500
GL500
GL520
CAS No: 1571954-81-8
Action of test item: Plasticizer
Appearance/Physical state: Clear colorless liquid
Batch: GLFG160607
Purity: 99.8%
Expiry date: 07 June 2017
Storage conditions: Room temperature in the dark
Specific details on test material used for the study:
Test item: GL500
Test item identity (including alternative names): LGFlex GL500, GL500, GL520
CAS number: 1571954-81-8
Intended use: Plasticizer
Appearance : Clear colorless liquid.
Storage conditions: At ambient temperature (15 to 25°C) and protected from light (although may be used and formulated in light).
Supplier: Sponsor
Batch number: GLFG160607
Expiry date: 1 June 2017.
Purity: 99.8%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken and placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST
Details on test animals or test system and environmental conditions:
Animals
Strain/Species RccHan™;WIST rat.
Supplier Envigo RMS Limited.
Number of animals ordered 90 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Six days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 78 to 84 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 181 to 214 g.

Animal Care and Husbandry
Environmental Control
Rodent facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Acclimatization up to four animals
During pairing one (stock) male and one female
Gestation one female

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The study consisted of one control and three treated groups identified as follows:
Group Treatment Dose# Number of animals Animal numbers
(mg/kg/day) Female Female
1 Control 0 20 1-20
2 GL500 100 20 21-40
3 GL500 330 20 41-60
4 GL500 1000 20 61-80
# Expressed in terms of material as supplied

Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
(On one day there was a lag in dosing time from the previous day of more than ± 2 hours. Refer to Section 4 of this report for further information).
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Procedure
The samples were analyzed in accordance with the validated Envigo Analytical Procedure Method No.DFA/M116/16.
The analytical methodinvolved extraction in acetoneand dilution inacetonitrile/water 90/10v/v followed by reverse phase high performance liquid chromatographic analysis with ultra violet detection at 241 nm. Sample concentrations were determined with reference to external standards prepared in the concentration range 0.5 µg/mLto 5.0 µg/mL

Concentration of Dose Formulations
The formulations for Week 1(GD6) and the Final Week(GD19)of treatment were sampled. For Groups 2, 3 and 4, 4 × 1mL (accurately weighed) was sampled from the middle of the formulation by Pharmacy personnel. For Group1, 2 × 3 mL (accurately weighed) was sampled from the middle of the formulation by Pharmacy personnel.
Two samples from Groups 2, 3 and 4 and duplicate aliquots from one Group 1 sample were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.
Re-dilution of the original results and contingency sample analysis was performed for Group 3MF (GD6 and GD19) to confirm a low result. This was confirmed to be a dilution error in the initial analysis and the mean of all 4 results of reported.

RESULTSAND CONCLUSION
The mean concentrations were within ±3% of the nominal concentration, confirming the accuracy of formulation. The % difference from mean (or coefficient of variation for Group 3MF) was found to be within 5% confirming precise analysis
Details on mating procedure:
Mating
Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day
Frequency of treatment:
Females were treated once daily at approximately the same time each day
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled body weight
Dose / conc.:
330 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled body weight
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Calculated from the most recently recorded scheduled body weight
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
The study consisted of one control and three treated groups identified as follows:
Group Treatment Dose# Number of animals Animal numbers
(mg/kg/day) Female Female
1 Control 0 20 1-20
2 GL500 100 20 21-40
3 GL500 330 20 41-60
4 GL500 1000 20 61-80
# Expressed in terms of material as supplied

Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
(On one day there was a lag in dosing time from the previous day of more than ± 2 hours. Refer to Section 4 of this report for further information).
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Examinations

Maternal examinations:
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing
Detailed observations were recorded daily at the following times in relation to dose administration:
Pre-dose observation.
One to two hours after completion of dosing of all groups.
As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.
Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.
Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Terminal Investigations

Method of Kill
Method of kill for all adult animals Carbon dioxide asphyxiation.
Method of kill for fetuses Chilling on a cool plate (approximately 0C)

Necropsy
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.
Schedule Animals were killed on Day 20 after mating.
Sequence To allow satisfactory inter-group comparison.

Reproductive Assessment
The following were recorded for all animals:
Uterus Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).
Apparently non pregnant animals and for apparently empty uterine horns The number of uterine implantation sites was checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, E, 1964)).
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter Sexed internally and eviscerated.
Fixation Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid.
Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.
Statistics:
See below

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs observed in association with the dosing procedure and no signs at routine physical examination that were considered to be related to treatment. There were no deaths.
Slight piloerection was seen in one female receiving 1000 mg/kg/day (Animal No. 67) on Day 12 after dosing. Piloerection was also seen in one female receiving 330 mg/kg/day (Animal No. 56) and this animal was placed on special attention on Days 14 to 17 due to low food consumption and body weight loss. This animal was subsequently found to be not pregnant.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight gain (GD 6 - 20) for females treated at 1000 mg/kg/day was slightly low (87 %) when compared with Control. Overall body weight gains for females receiving 100 or 330 mg/kg/day were similar to those of the Control and were considered unaffected by treatment.
The adjusted mean body weight gain (Days 6-20, adjusted for gravid uterine weight) was markedly low in females receiving 1000 mg/kg/day (63% of Control: a gain of 17 grams for females given 1000 mg/kg/day compared with a gain of 27 grams for the Control female group).
Mean gravid uterine weights were generally similar to Control at 100, 330 or 1000 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The overall food consumption during gestation (Days 6-19) was similar to Control at 100, 330 or 1000 mg/kg/day.
Food consumption was marginally low in females receiving 1000 mg/kg/day on Days 6-9 (88% of Control) and Days 18-19 (89% of Control) of gestation
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings, considered to be related with treatment, in the adults.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
One control female 1F 09 and one female receiving either 100 mg/kg/day (2F 23) or 330 mg/kg/day (3F 56) were found to be not pregnant, therefore there were 19, 19, 19 and 20 females at 0, 100, 330 or 1000 mg/kg/day respectively, with live fetuses at necropsy for evaluation.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were considered to be no test item related effects on numbers of implantations, resorptions (early or late), live young, sex ratio and pre or post-implantation loss.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were considered to be no test item related effects on numbers of implantations, resorptions (early or late), live young, sex ratio and pre or post-implantation loss.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
330 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on placental, litter or fetal weights in animals receiving GL500 at doses up to 1000 mg/kg/day.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no major fetal abnormalities related to treatment.
At 330 and 1000 mg/kg/day there was an increased incidence of supernumerary 14th ribs (minor skeletal abnormality), compared with the concurrent Control, but the incidences were within the HCD and were therefore considered to be unrelated to treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg/day there was also an increased incidence of brain haemorrhages compared with concurrent Control (recorded in six fetuses compared with two fetuses with this finding in the Control group). However, similar incidences of brain haemorrhages were evident in the HCD and therefore this finding was also considered to be unrelated to treatment.
There was some evidence of ossification delays in the fetuses of females given 1000 mg/kg/day. When compared with the Control, such findings included an increased incidence of delayed/incomplete ossificiation/unossified cranial bones and 5th/6th sternebrae and also a decrease in the incidence of ossified cervical vertebral centra. The incidences of these findings were outside the HCD. In addition, when compared with the Control, there was an increased incidence of partially undescended thymus and this finding was also outside the HCD. Ossification delays are a transient stage in fetal development and are not considered adverse. They are, together with the finding reported in the thymus, indicative of a slight delay in fetal development.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
skeletal malformations
other: Other effects

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
It is concluded from this study that the dosage of GL500 at 330 mg/kg/day was the maternal no-observed-adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no observed adverse-effect-level (NOAEL) for embryo-fetal survival and development.
Executive summary:

The purpose of this study was the assessment of the influence of GL500, a plasticizer, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in theHan Wistar rat.

Three groups of 20 females received GL500 at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at thesame volume dose as treated groups and for the same duration. Animals were killed on Day 20 of mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

Maternal clinical condition, body weight, food consumption and macroscopic evaluation were not adversely affected by treatment with GL500 at doses of 100 or 330 mg/kg/day. At 1000 mg/kg/day, there was a reduction in overall mean body weight gain (87% of Control) and also the adjusted mean body weight gain (adjusted for gravid uterine weight) was reduced (63% of Control).  At1000 mg/kg/day, mean food consumption was reduced during Days 6-9 and 18-19 of gestation.

There wasno effect on mean gravid uterine weight and adults were macroscopically normal. 

The number of implantations (including pre or post-implantation loss), resorptions (early or late), live young, sex ratio of males to females and placental and fetal weights were unaffected by treatment.

The pathological examination of the fetuses revealed, when compared with the Controls, higher incidences of the minor findings of delayed ossification of some fetal bones and undescended thymus at 1000 mg/kg/day. These effects were considered to represent a transient stage in development and not to be detrimental to the growth, development and survival of the fetuses.

Conclusion

It is concluded from this study that the dosage of GL500 at 330 mg/kg/day was the maternal no-observed-adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no‑observed‑adverse-effect-level (NOAEL) for embryo-fetal survival and development.