Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2018 - 22 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Pale yellow solid block
Details on test material:
Identification: NPG-THPA Hemiester (TK12094)
Description: Pale yellow solid block
Batch: Colkb03
Purity: 96%
Expiry / Retest Date: 01 February 2021
Storage Conditions: Room temperature in the dark over silica gel
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: Colkb03
- Expiration date of the batch: 01 February 2021
- Purity: > 85 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature over silica gel in darkness

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 224 to 323g.
- Housing: Solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20 %
- Air changes (per hr): 15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 22 Jun 2018 To: 11 July 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of the study, the test item was prepared at the appropriate concentrations in Propylene glycol. The stability and homogeneity of the test item formulations were determined. Bulk formulations were therefore prepared one occasion, divided into daily aliquots and stored at approximately 4 °C in the dark until the day of use.

VEHICLE
- Concentration in vehicle: 0, 20, 60, 150 mg/mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analyzed for concentration of 2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate. The results indicate that the prepared formulations were within 100 to 103% of the nominal concentration and were acceptable for use on the study.
Frequency of treatment:
Daily
Duration of test:
from Day 3 to Day 19 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on previous toxicity data available toxicity data, including data from a Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat and a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat.

- Other: The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: On days 3, 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Kidney, Ovaries, Uteri
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, mean food consumption was statistically significantly lower than control from the start of treatment (Day 3 of gestation) until Day 8 of gestation. Thereafter, food intake during gestation was considered to be similar to control until termination on Day 20 of gestation.

There was no effect of treatment on food consumption during gestation at 100 or 300 mg/kg bw/day.
Gross pathological findings:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food efficiency
gross pathology

Results (fetuses)

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
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on these findings of this pre-natal study, a dosage of 750 mg/kg bw/day was regarded as a No Observed Adverse Effect Level (NOAEL) for the pregnant female.
The findings observed for fetuses at 750 mg/kg bw/day were considered to be unrelated to treatment and this dosage of 750 mg/kg bw/day was considered to represent at least a NOAEL, and most probably a No Observed Effect Level (NOEL), for the survival, growth and development of the conceptus.
Executive summary:

Introduction

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated daily administration by gavage to the pregnant female during Days 3 to 19 of gestation, including the period of organogenesis.

The study was designed to comply with the following guidelines:

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 Nousan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels of 100, 300 and 750 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Propylene Glycol) over the same treatment period to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results…….

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

Clinical signs did not indicate any systemic effect of treatment at 100, 300 or 750 mg/kg bw/day.

Body Weight

At 750 mg/kg bw/day, mean body weight gain was statistically significantly lower than control on the first day of treatment, thereafter body weight gain was unaffected by treatment and mean overall body weight gain at termination, including when adjusted for the contribution of the gravid uterus, was similar to control.

There was no effect of treatment on body weight gain during gestation, including when values were adjusted for the contribution of the gravid uterus, at 100 or 300 mg/kg bw/day. 

Food Consumption

At 750 mg/kg bw/day, mean food consumption was statistically significantly lower than control from the start of treatment to Day 8 of gestation. 

There was no effect of treatment on food consumption during gestation at 100 or 300 mg/kg bw/day. 

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies

Macroscopic necropsy findings observed for adult animals did not indicate any effect of treatment at 100, 300 or 750 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

There was no effect of maternal treatment on pre-implantation loss, number of implantations, subsequent embryofoetal survival, litter size or sex ratio on Day 20 of gestation at 100, 300 or 750 mg/kg bw/day.

Fetal Examination

External Examination

The type, incidence and distribution of external findings at 100, 300 and 750 mg/kg bw/day did not indicate any effect on fetal development, although it was noted that one fetus at 300 mg/kg bw/day showed omphalocoele (portion of protruding intestine) and one fetus at 750 mg/kg bw/day showed spina bifida (lower spine exposed).

Visceral Examination

The type, incidence and distribution of visceral findings at 100, 300 and 750 mg/kg bw/day did not indicate any obvious effect on fetal development. The previous external findings of one fetus at 300 mg/kg bw/day showing omphalocoele (portion of protruding intestine) and one fetus at 750 mg/kg bw/day showing spina bifida (lower spine exposed) were confirmed. 

Skeletal Examination

The type, incidence and distribution of skeletal findings at 100, 300 and 750 mg/kg bw/day did not indicate any adverse effect on fetal development.

At 750 mg/kg bw/day, the incidences of fetuses showing incomplete ossification of a number of skull bones (nasal, frontal, parietal, interparietal, squamosal, jugal, zygomatic process of maxilla, zygomatic process of maxilla and hyoid) were statistically significantly lower than control. Additionally statistically significant lower incidences of incomplete ossification of the sacral (neural) arch, pubis and metacarpels were also apparent and there was a statistically significant higher incidence of foetuses with one or more ossified forepaw phalanges at this dosage. 

At 300 mg/kg bw/day, statistically significant differences from control in the incidence of skeletal findings were restricted to lower incidences of fetuses showing incomplete ossification of the parietal, jugal, zygomatic process of maxilla, sacral (neural) arch and pubis. 

At 100 mg/kg bw/day, the incidences of fetuses showing incomplete ossification of the hyoid and pubis were statistically significantly lower than control.

 

Conclusion

Based on these findings of this pre-natal study, a dosage of 750 mg/kg bw/day was regarded as a No Observed Adverse Effect Level (NOAEL) for the pregnant female.

The findings observed for fetuses at 750 mg/kg bw/day were considered to be unrelated to treatment and this dosage of 750 mg/kg bw/day was considered to represent at least a NOAEL, and most probably a No Observed Effect Level (NOEL), for the survival, growth and development of the conceptus.