Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21-Oct-2014 to 24-Apr-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 12-Aug-2014 to 17-Sep-2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22nd January 2001
Deviations:
yes
Remarks:
- reduced examination and number of test animals (only 8 per dose group instead of the required 20)
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: At least 11 weeks at day 0 post coitum
- Weight at study initiation: 206 - 258 g at day 0 post coitum
- Fasting period before study: None
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands).
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 13/14) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 12-Aug-2014 (start of 7-day Acclimatization) To: 17-Sep-2014 (Last Caesarean Section and Necropsy)
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
300
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test item as supplied by the Sponsor.
Vehicle was pre-warmed to a temperature of approximately 40 °C. DGEBADA was weighed into a glass beaker on a tared precision balance and approximately 80 % of the warm vehicle was added (w/v). The formulations were mixed with a magnetic stirrer until a homogeneous solution was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume and thereafter stirred for approximately 15 minutes. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

DIET PREPARATION
- Not relevant due to exposure via gavage

VEHICLE
- Justification for use and choice of vehicle: Considered non-toxic to the test animals and ability to form a homogeneous mixture with the test item
- Concentration in vehicle (nominal): 0, 25, 75 and 250 mg/mL (at 0, 100, 300 and 1000 mg/kg bw/day, respectively)
- Amount of vehicle (by gavage): 4 mL/kg body weight with a daily adjustment to the actual body weight
- Lot/batch no.: BCBK9989V (Source: Sigma-Aldrich Chemie GmbH, Steinheim / Germany, Expiry Date: 20-May-2016)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item was used as the analytical standard.
Concentration and homogeneity of dose formulations were determined in samples taken after experimental start and on the last day of treatment.
Transport of Dose Formulations to Analytical Laboratory: At ambient temperature (20 ± 5 °C)
Storage of Dose Formulations in Analytical Laboratory: Frozen (ca. -20 ± 5 °C).
The samples (approximately 1 g each) were delivered to the analytical laboratory and analyzed using method DGEBADA_PEG300 following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The method is described in detail in fully OECD TG 414 compliant prenatal developmental toxicity test of Braun 2015 (Harlan Laboratories no. D84008).

Two formulations made at 26-Aug-2014, i.e. Day 6 and one day before the last administration at 16-Sep-2014, i.e. Day 27 were analysed.

Dose group 1 (0 mg/kg bw/day corresponding to 0 mg/mL vehicle PEG 300):
- No test item could be detected in both analysed formulations.

Dose group 2 (100 mg/kg bw/day corresponding to 25 mg/mL vehicle PEG 300):
- formulation of 26-Aug-2014:
Mean recovery 82.4 % (in the samples from top, middle and bottom 82.2, 82.1 and 82.8 %, respectively);
coefficient of variation 0.4 %
- formulation of 16-Sep-2014:
Mean recovery 94.0 % (in the samples from top, middle and bottom 92.8, 95.4 and 93.8 %, respectively);
coefficient of variation 1.4 %

Dose group 3 (300 mg/kg bw/day corresponding to 75 mg/mL vehicle PEG 300):
- formulation of 26-Aug-2014:
Mean recovery 73.6 % (in the samples from top, middle and bottom 73.9, 73.5 and 73.6 %, respectively);
coefficient of variation 0.3 %
- formulation of 16-Sep-2014:
Mean recovery 97.4 % (in the samples from top, middle and bottom 97.3, 96.4 and 98.6 %, respectively);
coefficient of variation 1.1 %

Dose group 4
- formulation of 26-Aug-2014:
Mean recovery 80.2 % (in the samples from top, middle and bottom 80.3, 80.3 and 80.1 %, respectively);
coefficient of variation 0.1 %
- formulation of 16-Sep-2014:
Mean recovery 91.4 % (in the samples from top, middle and bottom 88.9, 93.1 and 92.3 % , respectively);
coefficient of variation 2.5 %
Details on mating procedure:
- Impregnation procedure: Cohoused; After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- M/F ratio per cage: 1:1
- Length of cohabitation: After 8 days pairing of all test animals was successfully completed.
- Further matings: No
- Verification of same strain and source of both sexes: Yes, all animals belonged to the RccHan™: WIST(SPF) strain
- Proof of pregnancy: Vaginal plug observed or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: None
Duration of treatment / exposure:
15 days (during gestation period from day 6 to 20 post coitum)
Frequency of treatment:
Daily, at approximately 24 hour intervals
Duration of test:
21 days post coitum
Experimental Starting Date (Delivery of Animals): 12-Aug-2014
Acclimatization: 7 days from 12-Aug-2014 (Day -8)
Initiation of Pairing: 19-Aug-2014 (Day -1)
First Test Item Administration: 26-Aug-2014 (Day 6)
First Caesarean Section and Necropsy: 10-Sep-2014 (Day 21)
Termination (Last Caesarean Section and Necropsy): 17-Sep-2014 (Day 28)
Experimental Completion Date: 17-Sep-2014 (Day 28)
No. of animals per sex per dose:
Each group consisted of 8 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous Combined 4-Week General Toxicity and Reproductive Developmental Toxicity Screening Study in the Rat with DGEBADA (Huntingdon Life Science study no. RAJ002).
- Rationale for animal assignment: Computer-generated random algorithm
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (once daily, during acclimatization and up to day of necropsy)
- Cage side observations were included in the effect level remarks fields

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of foetuses in the uterus was performed and the data recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain. Gravid uterus weights were recorded. The foetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, killed by injection of sodium pentobarbital and discarded.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions (embryonic): Yes
- Number of late resorptions (foetal): Yes
- Other: If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulphide to accentuate possible haemorrhagic areas of implantation sites
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
The following statistical methods were used to analyse food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied, if the variables could be dichotomized without loss of information.
Indices:
No indices were calculated as percentages were used.
Historical control data:
In absence of effects historical control data were not used for the interpretation of the results.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In groups 3 and 4, bedding material in the mouth was observed in all animals from day 9 post coitum at the earliest until the end of the study.
No clinical signs were observed in group 2.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females survived the scheduled study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on body weight development were recorded.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption were recorded.
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
During macroscopic examination no changes were noted in any dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
No effects of the treatment with the test item on reproduction data were recorded. All animals except one group 3 female were pregnant. This was not considered to be a test item-related effect, because all females were pregnant at the high dose (group 4). In group 4, the pre-implantation loss (relative to the number of corpora lutea) and the number of implantation sites (relative to the number of corpora lutea) were statistically significant decreased. These effects were not test item-related, because the implantation takes place before the treatment start on day 6 post coitum.
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
- External Abnormalities and Variations: No findings were noted during external examination of the foetuses.
- Sex Ratios: No effects on the sex ratio of the foetuses were noted.
- Body Weights: No effects of the treatment with the test item on foetus weights were recorded.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
not specified

Maternal Data:

Table 1: Summary of Performance of Mated Females

Group
Dose (mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

1 - 8

9 - 16

17 - 24

25 - 32

Number of mated females

8

8

8

8

Pregnant (A)

8

8

7

8

Number of females with live fetuses at termination*

8

8

7

8

* Only dams with at least one live fetus at Caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data.

(A) Females no. 17 was not pregnant.

Conclusions:
Based on the results of this study, dose levels of 0, 100, 300 and 1000 mg/kg bw/day are considered to be suitable for the subsequent main study for effects on embryo-foetal development in the Han Wistar rat.
Executive summary:

The oral (gavage) prenatal developmental toxicity of the test item 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid (CAS 55818-57-0, DGEBADA) to Wistar rats was investigated in a non-GLP dose range-finding study similar to the OECD TG 414 (2001) protocol.

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 8 mated females per group were treated by gavage with DGEBADA once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). The analytical verification revealed in the first analysed formulation 82.4, 73.6 and 80.2 % of the nominal values for groups 2, 3 and 4, respectively and in the second one higher recovery levels of 94.0, 97.4 and 91.4 %, respectively.

Using PEG 300 as vehicle a standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was administered. Control animals were dosed with the same volume of vehicle alone.

All females survived the scheduled study period. In groups 3 and 4, bedding material in the mouth was observed in all animals from day 9 post coitum at the earliest until the end of the study. No clinical signs were observed in group 2. In all groups no effects on food consumption, no effects of the treatment with the test item and no effects of the treatment with the test item on reproduction data were recorded. During macroscopic examination no changes were noted in any dose group.

During external examination of foetuses no external abnormalities and variations, no effects on the sex ratio and no test item-related effects on foetal body weights were noted.

In conclusion based on the results of this study, nominal dose levels of 0, 100, 300 and 1000 mg/kg bw/day are considered to be suitable for the subsequent main study for effects on embryo-foetal development in the Han Wistar rat.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22nd January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
EC 2004/73 B31, dated April 29, 2004
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
EPA 712-C-98-207, dated August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: At least 11 weeks at day 0 post coitum
- Weight at study initiation: 173 to 261 g at day 0 post coitum
- Fasting period before study: None
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands).
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 13/14) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed.
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 21-Oct-2014 (start of 7-day Acclimatization) To: 27-Nov-2014 (Last Necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
300
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared at least once weekly using the test item as supplied by the Sponsor.
Vehicle was pre-warmed to a temperature of approximately 40 °C. DGEBADA was weighed into a glass beaker on a tared precision balance and approximately 80 % of the warm vehicle was added (w/v). The formulations were mixed with a magnetic stirrer until a homogeneous mixture was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume and thereafter stirred for approximately 15 minutes. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Stability and Storage of Dose Formulations: For at least 10 days at room temperature (20 ± 5 °C) based on stability results of Harlan Laboratories Study no. D94595 (Braun 2015, subchronic repeated oral toxicity

DIET PREPARATION
- Not relevant due to exposure via gavage

VEHICLE
- Justification for use and choice of vehicle: Considered non-toxic to the test animals and ability to form a homogeneous mixture with the test item
- Concentration in vehicle (nominal): 0, 25, 75 and 250 mg/mL (at 0, 100, 300 and 1000 mg/kg bw/day, respectively)
- Amount of vehicle (by gavage): 4 mL/kg body weight with a daily adjustment to the actual body weight
- Lot/batch no.: BCBK9989V (Source: Sigma-Aldrich Chemie GmbH, Steinheim / Germany, Expiry Date: 20-May-2016)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples were dispatched to the analytical laboratories internally (at room temperature) and stored frozen at -20 ± 5 °C until analysis.
The test item was used as the analytical standard.
Stock solutions of DGEBADA in acetonitrile were prepared for external calibration. For example, 31.12 mg of DGEBADA was weighed into a 50 mL volumetric flask and filled to about 75 % of final volume with acetonitrile. The mixture was sonicated for at least five minutes and brought to volume with acetonitrile to yield a solution with a concentration of 622.4 µg/mL. Aliquots of this stock calibration solution were diluted with acetonitrile to obtain calibration solutions with nominal concentrations ranging from 10.58 to 200.4 µg/mL. On each occasion calibration solutions derived from two stock solutions were used for calibration.
Each sample was transferred into an appropriate volumetric flask. The sample vial was successively rinsed with at least two portions of acetonitrile and the rinsings were combined in the volumetric flask. The flask was filled to about 75 % of the target volume with acetonitrile and dissolution was achieved by sonication for at least five minutes. The flask was filled to the mark with acetonitrile. Where necessary, sample solutions were further diluted with acetonitrile into the calibration range.
Typical HPLC systems from Merck-Hitachi series 7000 were used:
- Computerized System: EZ Chrom Elite; Agilent Technologies
- Column: Kinetex C18; 50 x 4.6 mm, 5 µm
- Pre-Column: Phenomenex C18; 4 x 3 mm
- Column Temperature: Ambient
- Eluent A: Purified water / acetonitrile (50/50, v/v)
- Eluent B: Acetonitrile
- Gradient:
Time [min]; Eluent A [%]; Eluent B [%]
0; 100; 0
4; 100; 0
8; 5; 95
10; 5; 95
10.1; 100; 0
15; 100; 0
- Flow: 1 mL/min
- Wave Length: 230 nm
- Injection Volume: 10 µL
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20 % variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R²) were higher than 0.99.
The DGEBADA concentrations in the analysed dose formulations ranged from 86.0 to 102.0 % (dates of Preparation of the stock solution 03-Nov-2014, Day 6 and 14-Nov-2014, Day 17) with reference to the nominal and were within the accepted range of ±20 %. The homogeneous distribution of DGEBADA in the preparations was approved because single results found did not deviate more than 7.3 % from the corresponding mean and met the specified acceptance criterion of =15 %. Accordingly the effects were assigned to the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: Cohoused, After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- M/F ratio per cage: 1:1
- Length of cohabitation: After 10 days pairing of all test animals was successfully completed.
- Further matings: No
- Verification of same strain and source of both sexes: Yes, all animals belonged to the RccHan™: WIST(SPF)
- Proof of pregnancy: Vaginal plug observed or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: None
Duration of treatment / exposure:
15 days (during gestation period from day 6 to 20 post coitum)
Frequency of treatment:
Daily, at approximately 24 hour intervals
Duration of test:
21 days post coitum
Experimental Starting Date (Delivery of Animals): 21-Oct-2014
Acclimatization: 6 days from 21-Oct-2014 (Day -7)
Initiation of Pairing: 27-Oct-2014 (Day -1)
First Test Item Administration: 03-Nov-2014 (Day 6)
First Necropsy (Caesarean Section): 18-Nov-2014 (Day 21)
Termination (Last Necropsy): 27-Nov-2014 (Day 30)
Experimental Completion Date: 24-Apr-2015
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding study in Han Wistar rats, Harlan Laboratories non-GLP study no. D83997, using dose levels of 0, 100, 300 and 1000 mg/kg/day.
- Rationale for animal assignment: Computer-generated random algorithm

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Viability / Mortality observation were made twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
- Time schedule: For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: Gross macroscopic examination of all internal organs
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Post mortem examination with emphasis on the uterus, uterine contents, corpora lutea count and position of foetuses in the uterus was performed and the data recorded.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions (embryonic): Yes
- Number of late resorptions (foetal): Yes
- Other: The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Foetuses were removed from the uterus by caesarean section, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. Microdissection technique (sectioning/dissection technique): Approximately one half of the foetuses from each litter were fixed in Bouin's fixative. They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
2. The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol, carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.

The foetuses were sent to the contributing scientist for peer review at Huntingdon Life Sciences.
Statistics:
The following statistical methods were used to analyse food consumption, body weights, reproduction and skeletal examination:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied, if the variables could be dichotomized without loss of information.
Indices:
No indices were calculated as percentages were used.
Historical control data:
Tables summarizing the available historical control data from six studies performed at the test site (referenced 12/02, 12/03, 12/04, 13/01, 13/02 and 13/03) are given in Appendix V of the study report.
For the interpretation of results regarding Ribs (Supernumerary cervical, Rudimentary) the historical data record was used. It showed occurrences in studies 12/02, 12/04, 13/01 and 13/02, while such effects lacked in study 12/03 and were not examined in study 13/03 (data shown on page 329 of the study report):
In foetuses the numbers and the related percentages were: 1 (1 %), 1 (1 %), 1 (1 %) and 3 (2 %)
In litters the numbers and the related percentages were: 1 (5 %), 1 (4 %), 1 (5 %) and 2 (10 %)
Furthermore foetal body weight gain and non-ossification data were used for data interpretation of this study, but they were due to their complexity not reproduced here.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical symptoms or signs were observed at any dose during the study. Female no. 54 (group 3) had vaginal bleeding, showed ruffled fur and decreased activity, and was in a general weakened condition on day 20 post coitum. These symptoms were not considered to be test item-related, because they were restricted to this single animal in this group and no clinical signs were observed in animals of group 4.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female no. 54 (group 3) was sacrificed on day 20 post coitum. The animal had vaginal bleeding, showed ruffled fur and decreased activity, and was in a general weakened condition. All other females survived until the scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No effects on mean absolute body weights were recorded. T
ransiently statistically significant changes in body weight gain were observed in the group 2 on Day 14, and in the group 4 on Day 9 and from Day 12 to 16; there were of minimal magnitude and considered to be incidental findings.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily food consumption was similar in all groups from days 0-6 post coitum. There were no test item-related changes in food consumption during days 6-21 post coitum.
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 specified
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related findings were observed at any dose level during macroscopic examination. Pale discoloured lungs which were reduced in size, spleen reduced in size, a clay-coloured liver, and a haemorrhagic-watery fluid containing stomach were recorded in female no. 54 (group 3) which was sacrificed on day 20 post coitum in moribund condition.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):

Placenta Weights:
In group 4, placenta weights on a litter basis were increased by 23 and by 24 % on an individual basis. This increase was considered to be test item-related. Slight, but statistically increased placenta weights on an individual basis in groups 2 and 3 were not considered to be test item-related, because the increase was only minor (+8 and +6.5 %, respectively) and not dose-related.
Mean placenta weights calculated on a litter basis were: 0.484 g, 0.515 g, 0.510 g and 0.594 g whereas calculated on an individual basis, they were 0.476 g, 0.514 g, 0.507 g and 0.589 g, both cited in order of ascending dose level.
Details on maternal toxic effects:
The statistically significant decrease in the number of implantation sites and a decrease of the pre-implantation loss in all test item-treated groups were not test item-related since this occurs before treatment starts.
The relevant reproduction data (mean post-implantation loss and mean number of foetuses per dam) were not affected by treatment with the test item.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In group 4, placenta weights on a litter basis were increased by 23 and 24 % on litter and individual bases, respectively, and considered to be test item-related. Differences noted in groups 2 and 3 were unrelated to dose and therefore considered to be incidental.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No test item-related effects on mean body weights of live foetuses were recorded.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratios were similar to those of the controls.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No test findings were observed during external examination of the foetuses.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test item-related findings were noted during external and fresh visceral examinations of foetuses or during the skeletal examination of the foetuses.
The incidence of incomplete or non-ossification was slightly elevated in a small number of bones: Most of these values remained within the ranges of the respective historical control values. A small number of supernumerary ribs remained within the ranges of the respective historical control values and are therefore considered to be of no toxicological relevance.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test item-related findings were noted during external and fresh visceral examinations of foetuses or during the skeletal examination of the foetuses.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
No test item-related findings were noted during external and fresh visceral examinations of foetuses or during the skeletal examination of the foetuses.In some cases, these values exceeded the historical control data ranges but were considered to be specific structures that typically vary in development. These findings are unlikely to be related to the treatment and therefore considered to be of no toxicological relevance.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion based on these results, the NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be above 1000 mg/kg bw/day. Placenta weight differences seen in group 4 were considered to be test item-related changes; the NOEL for prenatal development was therefore considered to be 300 mg/kg/day. The NOAEL for prenatal developmental toxicity was defined as 1000 mg/kg bw/day.
Executive summary:

The oral (gavage) prenatal developmental toxicity of the test item 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid (CAS 55818-57-0, DGEBADA) to Wistar rats was investigated in a GLP-compliant dose-effect study according to the OECD TG 414 (2001).

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 24 mated females per group were treated by gavage with DGEBADA once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (PEG 300). All females were sacrificed on day 21 post coitum and the foetuses were removed by caesarean section.

Regarding the maternal data, no test item-related premature deaths were recorded. One female in group 3 was sacrificed for ethical reasons unrelated to the treatment with the test item. No test item-related clinical symptoms or signs, no changes in the food consumption and no effects on mean absolute body weights and body weight gain were observed at any dose during the study. The relevant reproduction data (post-implantation loss and number of foetuses per dam) were not affected by treatment with the test item and no test item-related macroscopic findings were observed at any dose level during macroscopic examination.

The foetal examinations revealed in group 4 placenta weights on a litter basis being increased by 23 and by 24 % on an individual basis. This increase was considered to be test item-related. Differences noted in groups 2 and 3 were unrelated to dose and therefore considered to be incidental. No test findings were observed during external examination of the foetuses. No test item-related effects on sex ratio and mean body weights of the foetuses were noted in any group. No test item-related abnormalities and variations were noted during external and fresh visceral examinations of foetuses and no findings were noted during skeletal examination of the foetuses. A small number of incomplete or non-ossified bones and/or supernumerary ribs remained within the ranges of the respective historical control values and were therefore considered to be of no toxicological relevance. There were no additional cartilage finding in any foetus that was considered to be related to the treatment with the test item.

In conclusion based on these results, the NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be above 1000 mg/kg bw/day. Placenta weight differences seen in group 4 were considered to be test item-related changes; the NOEL for prenatal development was therefore considered to be 300 mg/kg/day. The NOAEL for prenatal developmental toxicity was defined as 1000 mg/kg bw/day.