Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-21 to 2019-11-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147
Version / remarks:
24 November 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: 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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name of test material: Trimethylolpropanepoly(oxypropylene)triamine
EC no.: 500-105-6
CAS no.: 39423-51-3
Physical state: clear colourless liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3717
- Expiration date of the lot/batch: 01 December 2019
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services (under Envigo study number: QJ79QM). Results showed the formulations to be stable for at least twenty one days when stored at 4 °C in the dark.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Bulk formulations were prepared twice and then divided into daily aliquots and stored at approximately 4 °C in the dark for the 10, 100 and 200 mg/kg bw/day formulations. As the dose level was reduced from 200 to 125 mg/kg bw/day so close to the end of the study the 125 mg/kg bw/day formulations were prepared daily for the remainder of the study (07 July 2017 to 11 July 2017).

OTHER SPECIFICS:
No correction for purity was made.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD) IGS BR strain
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 96 time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation.
- Age at study initiation: no data
- Weight at study initiation: 172 to 300 g on arrival
- Fasting period before study: no data
- Housing: individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK)
- Diet (e.g. ad libitum): ad libitum, pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK)
- Water (e.g. ad libitum): ad libitum, mains drinking water from polycarbonate bottles attached to the cage
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting, 12 hours continuous light/12 hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Bulk formulations were prepared twice and then divided into daily aliquots and stored at approximately 4 °C in the dark for the 10, 100 and 200 mg/kg bw/day formulations. As the dose level was reduced from 200 to 125 mg/kg bw/day so close to the end of the study the 125 mg/kg bw/day formulations were prepared daily for the remainder of the study (07 July 2017 to 11 July 2017).

Treatment volume: 5 mL/kg

VEHICLE
- Concentration in vehicle: 2, 20, 40/25#
# dose level reduced from 200 mg/kg bw/day from Day 17
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) using an external standard technique. The test item gave a chromatographic profile consisting of two peaks. Samples were taken of each bulk test item formulation and the first daily preparation formulation (125 mg/kg bw/day) and were analyzed for concentration of the test substance.

Preparation of Calibration Standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration range of 0.05 mg/mL to 0.25 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters section.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.167 mg/mL by serial dilution covering the concentration range 0.05835 mg/mL to 0.3360 mg/mL.

Preparation of Test Samples
The formulations received were diluted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of Accuracy and Precision Samples
This was performed under study number QJ79QM.

Instrumentation Parameters
HPLC System : Agilent Technologies 1200 MSD, incorporating autosampler and workstation
Mass selective detector
Source: electrospray
Fragmentation energy: 70volts
Polarity: positive
Mode: single ion mode with 422.4 amu
Gas temperature: 350”C
Drying gas: 12.5 litre/minute
Nebuliser pressure: 40 psi
Capillary voltage: 2500 volts
Gain: 1
Column: Kinetix C8, 2.6 µ, (50 x 3 mm id)
Column temperature: 30”C
Gradient elution: eluent A: 0.1% formic acid in water
eluent B: acetonitrile
Time (minutes) % A) % B
0 100 0
5 20 80
10 100 0
Flow rate: 0.5 mL/min
Injection volume: 15.00 µL
Retention time: approximately 0.5 and 3.5 minutes

Data Evaluation and Calculations
The peak area response for the Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by non-linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

RESULTS
Concentration of Dose Formulations
The mean concentrations were within applied limits ±10%, confirming accurate formulation.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision during study number QJ79QM.
The homogeneity and stability was confirmed during study number QJ79QM.
The mean concentrations of Test Item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.
Details on mating procedure:
Pre-mated animals were ordered. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
16 days, from Day 3 to Day 19 of gestation
Frequency of treatment:
daily
Duration of test:
16 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group, vehicle only
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
high dose group, from start of treatment until day 17 of gestation
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
high dose group, from day 17 of gestation until end of treatment
No. of animals per sex per dose:
24 females/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen in collaboration with the Sponsor Representative and were based on available toxicity data including a Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat (Envigo Study Number KM11LR) and a 90 Day Oral (Gavage) Toxicity Study in the Rat (Envigo Study Number QJ79QM). 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.
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS
- Time schedule: once daily during the gestation period, immediately before and soon after dosing and one hour post dosing during the dosing period
- Parameters: overt signs of toxicity, ill-health or behavioral changes

BODY WEIGHT
- Time schedule: on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

FOOD CONSUMPTION
- Time schedule: at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION
- Time schedule: daily by visual inspection of the water bottles for any overt changes.

NECROPSY
- All surviving animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation.
- All animals were subjected to a full external and internal examination (including examination of the uterine contents) and any macroscopic abnormalities were recorded
Ovaries and uterine content:
- The ovaries and uteri of pregnant females were removed, examined and the following data recorded: Number of corpora lutea; Number, position and type of intrauterine implantation; Fetal sex; External fetal appearance; Fetal weight; Placental weight; Gravid uterus weight
- Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position.
Fetal examinations:
- The fetuses were killed by subcutaneous injection of sodium pentobarbitone.
- Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations.
- Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin.
- The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss:
Percentage pre-implantation loss was calculated as: [(number of corpora lutea - number of implantations)/number of corpora lutea] x 100

Percentage post-implantation loss was calculated as: ([number of implantations - number of live fetuses)/number of implantations] x 100

Sex ratio: % male fetuses (sex ratio) = (Number of male fetuses/Total number of fetuses) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were apparent in any animal during the course of the study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two animals treated with 200 mg/kg bw/day were found dead on the 07 July 2017 (Days 15 and 14 of gestation respectively). There were no clinical signs noted in either of these animals prior to death.
There were no further unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effect on body weight development was evident in animals from any treatment group.
Animals treated with 10 mg/kg bw/day showed a statistically significant increase (p<0.01) in cumulative body weight gain from Days 3 to 11 of gestation. An increase in body weight gain is considered not to reflect an adverse effect of treatment and was therefore considered not to be of toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effect on food consumption was evident in animals from any treatment group.
Animals treated with 200 mg/kg bw/day exhibited a statistically significant reduction (p<0.01) in food consumption from Days 3 to 5 of gestation. As recovery was evident thereafter and all food consumption data generated for this treatment group was within the historical control data range this reduction was considered not to be of any toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
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:
effects observed, treatment-related
Description (incidence and severity):
The two animals which were found dead on Days 15 and 14 of gestation exhibited fluid contents in the stomach and patchy pallor on the liver.
With the exception of one instance of yellow colored contents in the stomach in one animal treated with 200/125 mg/kg bw/day, no macroscopic abnormalities were detected in any surviving animal at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by pre- and post-implantation losses at 10, 100 or 200/125 mg/kg bw/day.
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by in utero offspring survival (as assessed by the mean numbers of early or late resorptions) at 10, 100 or 200/125 mg/kg bw/day.
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 10, 100 or 200/125 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
mortality

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Intergroup differences for mean fetal did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by sex ratio at 10, 100 or 200/125 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by live litter size at 10, 100 or 200/125 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.
Statistically significant reductions (p<0.05-0.01) in the number of fetuses exhibiting incomplete ossification of the hyoid bone of the skull, zygomatic process of squamosal of the skull and metacarpals were noted in animals treated with 10, 100 and 200/125 mg/kg bw/day, however, these were not regarded as evidence of developmental toxicity as these decreases were closer to the expected range for these parameters.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
125 mg/kg bw/day (nominal)
Treatment related:
no

Any other information on results incl. tables

Group Mean Litter Data Values

Dose Level (mg/kg bw/day)

 

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Implantation Loss
%

Number of Live Implants

%
Male
Fetuses

Mean Male Fetal Weight (g)

Mean Female Fetal Weight (g)

Mean Fetal Weight (g)

MeanPlacentalWeight
(g)

Litter Weight (g)

TotalPlacentalWeight
(g)

Early

Late

Total

Pre

Post

Male

Female

Total

0 (Control)

mean

16.8

13.4

0.2

0.0

0.3

19.9

1.9

6.9

6.2

13.1

52.7

4.115

3.882

4.012

0.582

52.594

7.590

sd

2.3

1.6

0.5

0.2

0.5

9.4

4.0

2.1

2.1

1.7

14.3

0.204

0.251

0.201

0.072

6.751

0.952

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

23#

10

mean

17.2

13.8

0.1

0.1

0.3

19.6

1.8

6.8

6.7

13.5

50.9

4.135

3.959

4.051

0.589

54.477

7.916

sd

2.5

2.3

0.4

0.4

0.6

10.6

4.6

2.6

2.6

2.3

16.5

0.276

0.282

0.248

0.066

8.989

1.502

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

100

mean

17.7

13.6

0.1

0.0

0.2

22.1

1.2

6.7

6.7

13.4

50.0

4.151

3.960

4.052

0.578

54.284

7.700

sd

2.7

1.5

0.3

0.2

0.4

11.5

2.7

2.0

1.9

1.4

14.1

0.247

0.234

0.224

0.090

5.662

1.121

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

200/125

mean

16.5

13.8

0.2

0.1

0.3

16.0

2.4

6.9

6.6

13.5

52.0

4.204

3.985

4.103

0.614

55.299

8.270#

sd

2.7

2.7

0.7

0.3

0.7

9.7

5.5

1.9

2.7

2.8

13.9

0.327

0.296

0.307

0.085

11.918

1.911

n

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

21

#= Total placental weight for Females 22 and 77 excluded from group mean as one placenta not weighed in error

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of JEFFAMINE T-403 to pregnant rats from gestation Days 3 to 19, at dose levels of 10, 100 or 200/125 mg/kg bw/day was associated with the deaths of two animals at 200 mg/kg bw/day. The No Observed Effect Level (NOEL) for the pregnant female was therefore considered to be 125 mg/kg bw/day as no further effects were apparent when the top dose level was reduced from 200 mg/kg bw/day to 125 mg/kg bw/day.
In-utero survival of the developing conceptus was unaffected by maternal treatment with 200/125 mg/kg bw/day. No changes in the measured fetal parameters or embryofetal development were detected at 10, 100 or 200/125 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be at least 125 mg/kg bw/day.
Executive summary:

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during 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 NohSan 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 10, 100, and 200 mg/kg bw/day (reduced to 125 mg/kg bw/day from Day 17 (animals 73-80), Day 16 (animals 81-88) or Day 15 (animals 89-96) of gestation onwards due to unscheduled mortality). Similar effects were also noted on a Ninety Day Repeated Dose study, Envigo Study Number: QJ79QM where dose levels were reduced from 200 to 150 mg/kg bw/day from Day 31 (males) and Day 30 (females), dose levels were subsequently reduced on this study again to 75 mg/kg bw/day from Day 36 (males) and Day 35 (females). A further group of twenty-four time mated females was exposed to the vehicle only (distilled water) 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 surviving 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

Two animals treated with 200 mg/kg bw/day were found dead on the 07 July 2017 (Days 15 and 14 of gestation respectively). There were no clinical signs noted in either of these animals prior to death.

There were no further unscheduled deaths.

Clinical Observations

No clinical signs were apparent in any animal during the course of the study.

Body Weight

No adverse effect on body weight development was evident in animals from any treatment group.

Food Consumption

No adverse effect on food consumption was evident in animals from any treatment group.

Water Consumption

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

Post Mortem Studies

The two animals which were found dead on Days 15 and 14 of gestation exhibited fluid contents in the stomach and patchy pallor on the liver.

With the exception of one instance of yellow colored contents in the stomach in one animal treated with 200/125 mg/kg bw/day, no macroscopic abnormalities were detected in any surviving animal at necropsy.

Litter Data and Litter Placental and Fetal Weights

There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations,in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 10, 100 or 200/125 mg/kg bw/day.

Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day.

Fetal Examination

External Findings

Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.

Detailed Visceral Examinations

Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.

Detailed Skeletal Examination

Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.

Conclusion

The oral (gavage) administration of JEFFAMINE T-403 to pregnant rats from gestation Days 3 to 19, at dose levels of 10, 100 or 200/125 mg/kg bw/day was associated with the deaths of two animals at 200 mg/kg bw/day.  The No Observed Effect Level (NOEL) for the pregnant female was therefore considered to be 125 mg/kg bw/day as no further effects were apparent when the top dose level was reduced from 200 mg/kg bw/day to 125 mg/kg bw/day. In-utero survival of the developing conceptus was unaffected by maternal treatment with 200/125 mg/kg bw/day. No changes in the measured fetal parameters or embryofetal development were detected at 10, 100 or 200/125 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be at least 125 mg/kg bw/day.