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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:
27 November 2012 - 08 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material: 4,4’-methylenebis[N,N-bis(2,3-epoxypropyl)aniline]
- Physical state: high viscous yellow to brown liquid
- Lot/batch No.: AAB0290400
- CAS Number: 28768-32-3
- Analytical purity: 84.9%
- Expiry date: 09 February 2014
- Storage conditions: at 5°C ± 3°C.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: 10-11 weeks old on the day of treatment
- Mean body weight at study initiation: 273 g (range: 223 g to 323 g)
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 4 or 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 03 December 2012 to 07 Jannuary 2013.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. It was mixed with the required quantity of vehicle according to the following process:
1. prepare a stock suspension at 200 mg/mL as detailed below:
- leave the test item at room temperature 30 min before preparation of the stock solution,
- weight the test item in a flask protected from light,
- add the vehicle,
- keep under continuous stirring at +37°C at least 2 hours and until a homogeneous preparation
is obtained (the time needed was recorded),

2. then, obtain the test item formulations by dilution as detailed below:
- weight an appropriate sample of the stock suspension in a flask protected from light,
- add the vehicle,
- stir at room temperature.
The test item dose formulations were prepared on a weekly basis. They were stored at +2-8°C and protected from light and delivered to the study room at room temperature and protected from light.

VEHICLE
- Justification for use and choice of vehicle: homogenous suspension in vehicle
- Concentration in vehicle: 0, 6, 18 and 54 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations (-5.1% to +0.9%): remained within an acceptable range of variation compared to nominal values (+/- 15%).
Stability: dose formulations were prepared on a weekly basis.
Details on mating procedure:
- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: vaginal plug at the breeder's facility.
Duration of treatment / exposure:
day 6 to day 20 post-coitum.
Frequency of treatment:
Daily
Duration of test:
27 November 2012 to 08 February 2013.
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 90 and 270 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a preliminary prenatal development study. In this study, three groups of five time-mated females were given the test item as a suspension in the same vehicle daily by gavage from days 6 to 20 p.c. at 50, 150 or 500/300 mg/kg/day. The high-dose group started the treatment with a dose-level of 500 mg/kg/day but was reduced to 300 mg/kg/day from day 10 p.c. following body weight losses and reduced food consumption especially. Another group of five time-mated females received the vehicle only and acted as a control group.

At 500/300 mg/kg/day, one pregnant female was prematurely sacrificed on day 16 p.c. for ethical reasons. In this group, test item-related clinical signs included: round back, piloerection, emaciated appearance, discolored feces and/or ptyalism. There was a mean body weight loss from day 6 to day 15 p.c. Mean body weight gain was then noted until the end of the study but it was lower than in controls. Mean body weight at the end of the gestation period was thus lower than in controls. Females had a mean net body weight loss from day 6 p.c. and thus a lower mean carcass weight than in controls. Mean food consumption was lower than controls during the whole treatment period, but more severe from days 6 to 15 p.c. At necropsy, 4/5 females (including the prematurely sacrificed female) showed thymus and spleen reduced in size. There were 2/5 females with at least three early resorptions vs. none in the control group, which resulted in higher mean post implantation loss.

At 150 mg/kg/day, the only test item-related clinical sign was ptyalism. Three out of five females had a body weight gain lower than the control range over the gestation period and two of them had a net body weight gain from day 6 p.c. lower than controls. Food consumption was slightly reduced for these three females in the period days 6 - 15 p.c. These three females also had a gravid uterus weight lower than controls.

At 50 mg/kg/day, there were no effects on mean body weights, mean food consumption, mean carcass weight and mean net body weight change from day 6 p.c. considered to be of toxicological importance. At hysterectomy, there were 2/5 females with at least three early resorptions vs. none in the control group resulting in higher mean post-implantation loss, and 3/5 females had a gravid uterus weight lower than controls.

The dose-level of 300 mg/kg/day was considered to be too high to be chosen as the high dose level in the main study because of the reduced food consumption (food consumption up to a maximum of ca.70% of the control group food consumption) and thus body weight gain.

- Rationale for animal assignment: computerized stratification procedure.

Examinations

Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on days 2, 4, 6, 9, 12, 15, 18 and 21 p.c., and prior to premature sacrifice.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on day 21 post-coitum.
- Examined: principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including::
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions
- Number of uterine scars,
- Evaluation of placenta
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: remaining fetuses
- Other : number dead and live, body weight, sex
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At 270 mg/kg/day, females had a mean gravid uterus weight lower than controls. The difference was statistically significant and considered to be adverse.
There were no relevant effects on mean gravid uterus weight at 30 or 90 mg/kg/day.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no effects of the treatment with the test item on fetal sex ratios.
At 270 mg/kg/day, mean fetal body weight (-20% from controls, p<0.001) and mean placenta weight (-27% from controls, p<0.001) were lower than in controls which correlated with the effect seen on mean gravid uterus. This was considered to be test item treatment-related and adverse. There were no effects on mean fetal body weight at 30 or 90 mg/kg/day or on mean fetal/placenta weight ratio at any dose-level.
There were no test item treatment-related external variations.
At 270 mg/kg/day, there was a trend toward an increase in incidences of soft tissue variations; an effect of the treatment with the test item could not be ruled out. There were also higher incidences than controls in the following findings: unossification and/or incomplete ossification of caudal vertebrae, 5 and 6th sternebrae and 1st metatarsal bone. There were no relevant soft tissue and skeletal variations at 30 and 90 mg/kg/day.
There was an increase in malformations at 270 mg/kg/day. In particular, there were 1 fetus in the 90 mg/kg/day group and 1 fetus in the 270 mg/kg/day group with thread-like tail and mainly absence of sacral and caudal vertebrae, plus of lumbar vertebrae at 270 mg/kg/day. A test item treatment-related effect cannot be excluded.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test item was administered by gavage, once daily, from days 6 to 20 p.c., inclusive, to time-mated female Sprague-Dawley rats at dosages of 30, 90 or 270 mg/kg/day.
On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 90 mg/kg/day based on the premature death, adverse clinical signs and effects on mean body weight and mean body weight change (including carcass and net body weight) and mean food consumption at 270 mg/kg/day,
- the NOAEL for embryo-fetal development was considered to be 90 mg/kg/day based on increased litter and fetal incidence of delayed/absent bone ossification and of malformations at 270 mg/kg/day in a context of severe maternal toxicity.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post-coitum (p.c.) inclusive).

Methods

Three groups of 24 time-mated Sprague-Dawley rats were administered the test item once daily from day 6 to day 20 p.c., by gavage at dosages of 30, 90 or 270 mg/kg/day. An additional group of 24 time-mated females received the vehicle, PEG 400, under the same experimental conditions and acted as the control group. A dose volume of 5 mL/kg/day was used.

 

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 or 3 days. On day 21p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.

 

Results

Chemical analyses

The test item concentrations in the administered dose formulations analyzed were within the acceptance criteria.

Pregnancy status

At termination on day 21 p.c., there were 22, 20, 19 and 18 dams with live fetuses in the vehicle control, 30, 90 and 270 mg/kg/day groups, respectively.

 

Mortality

One female treated at 270 mg/kg/day was prematurely sacrificed on day 20 p.c. for poor health condition. This death was considered to be test item treatment-related.

 

Clinical signs

At 270 mg/kg/day, round back (17/18 females), piloerection (17/18 females), emaciated appearance (7/18 females) and orange colored feces (3/18 females) mostly observed in the second half of the treatment period were considered to be related to the test item treatment and adverse (except for orange colored feces).

Ptyalism noted at 90 (2/19 females) and 270 (9/18 females) mg/kg/day was considered to be of minor toxicological significance.


Body weight, net body weight change and food consumption

At 270 mg/kg/day and when compared with controls, there were mean body weight losses or no mean body weight gain until day 15 p.c. and then lower mean body weight gain (mean body weight gain for the period of days 6-21 p.c.: -79% from controls, p<0.001). Mean body weights were lower from day 12 p.c. (mean body weight on day 2 1p.c.: -27% from controls, p<0.001).These effects correlated with lower mean food consumption throughout the treatment period (‑35 to -52%, p<0.001) and with a negative mean net body weight change from day 6 p.c. (vs.+, p<0.001) and lower mean carcass weight (-29%, p<0.001) at the end of gestation. These effects were considered to betest item-related andadverse.

At 90 mg/kg/day, there was a lower mean net body weight change from day 6 p.c.(+, p<0.01) and lower mean food consumption throughout the treatment period (from -6.7% to ‑11.1% from controls), both effects considered to be of minor toxicological significance. There was no relevant effect at 30 mg/kg/day on mean carcass weight and mean net body weight change or on mean body weight and mean body weight gain at 30 and 90 mg/kg/day.

 

Necropsy and hysterectomy data

There were no test item treatment-related findings at necropsy.

There were no effects on mean hysterectomy data (pre- and post‑implantation losses and early and late resorptions).

At 270 mg/kg/day, females had a mean gravid uterus weight lower than controls (-19%, p<0.01). This was considered to betest item treatment-related andadverse. There were no relevant effects on mean gravid uterus weight at 30 or 90 mg/kg/day.

 

Fetal examination

There were no effects of the treatment with the test item on fetal sex ratios.

At 270 mg/kg/day, mean fetal body weight (-20% from controls, p<0.001) and mean placenta weight (-27% from controls, p<0.001) were lower than in controls which correlated with the effect seen on mean gravid uterus. This was considered to betest item treatment-related andadverse. There were noeffects on mean fetal body weight at 30 or 90 mg/kg/day or on mean fetal/placenta weight ratio at any dose-level.

There were no test item treatment-related external variations.

At 270 mg/kg/day,there was a trend toward an increase in incidences of soft tissue variations; an effect of the treatment with the test item could not be ruled out.There were also higher incidences than controls in the following findings: unossification and/or incomplete ossification of caudal vertebrae, 5 and 6thsternebrae and 1stmetatarsal bone. There were no relevant soft tissue and skeletal variations at 30 and 90 mg/kg/day.

There was an increase in malformations at 270 mg/kg/day. In particular, there were 1 fetus in the 90 mg/kg/day group and 1 fetus in the 270 mg/kg/day group with thread-like tailand mainly absence of sacral and caudal vertebrae, plus of lumbar vertebrae at 270 mg/kg/day. A test item treatment-related effect cannot be excluded.


Conclusion

The test item was administered by gavage, once daily, from days 6 to 20 p.c., inclusive, to time-mated female Sprague-Dawley rats at dosages of 30, 90 or 270 mg/kg/day.

 

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 90 mg/kg/day based on the premature death, adverse clinical signs and effects on mean body weight and mean body weight change (including carcass and net body weight) and mean food consumption at 270 mg/kg/day,

.         the NOAEL for embryo-fetal development was considered to be 90 mg/kg/day based on increased litter and fetal incidence of delayed/absent bone ossification and of malformations at 270 mg/kg/day in a context of severe maternal toxicity.