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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2018 - January 2019
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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See details below
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See details below
Principles of method if other than guideline:
The following deviations from the study plan occurred:

- During the mating period of the preliminary phase, it was necessary to allocate two females which had only shown one copulation plug, although both showed 3+ (many scattered) sperm in the vaginal smear. These females were allocated as Group 1F No. 2 and Group 3F No. 41.

- Sections 5.2 and 16 of the study plan stated that the formulation samples collected during this study would be analysed by the Dose Formulation Analysis department at Envigo Huntingdon. This information was incorrectly entered in the study plan as the samples were analysed by the Residue Analytical Services department at Envigo Eye as it was always intended, where the formulation homogeneity and stability investigations were undertaken.

These deviations were considered not to have affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine
EC Number:
240-004-1
EC Name:
N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine
Cas Number:
15875-13-5
Molecular formula:
C18H42N6
IUPAC Name:
3,3',3''-(1,3,5-triazinane-1,3,5-triyl)tris(N,N-dimethylpropan-1-amine)
Test material form:
liquid: viscous
Details on test material:
Purity:
N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine
(CAS No. 15875-13-5) 98.0% (w/w)
Impurities:
Water (CAS No. 7732-18-5) 0.14 % (w/w)

Appearance: Colorless to pale yellow liquid
Batch No.: 1166046
Stability/Expiration: 06/2014
Specific details on test material used for the study:
Test item: N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine1,3,5(2H,4H,6H)-Tripropanamine
Appearance: Clear pale yellow liquid.
Storage conditions: Room temperature (15 to 25ºC) and stored in the dark.
Supplier: Sponsor.
Batch number: 2305905
Expiry date: 15 June 2020
Purity: 98.6%

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han Wistar (RccHan™ ;WIST) used because of the historical control data available at the test laboratory.
Details on test animals or test system and environmental conditions:
Strain/Species RccHan™;WIST rat.
Supplier Envigo RMS Limited.
Number of animals ordered: 92females in two deliveries (29 females for the preliminary phase and 63 females for the main phase). Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Preliminary phase: six days before commencement of pairing Main phase: five days before commencement of pairing.
Age of the animals at the start of the study (Day0 of gestation)
Preliminary phase: 78 to 84 days old Main phase: 77 to 83 days old.
Weight range of the animals at the start of the study (Day0 of gestation): 187to 222g

Rodent facility: Limited barrier - to minimizeentry of external biological and chemical agents and to minimizethe transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges. Lighting Artificial lighting, 12 hours light : 12hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose: 10mL/kg body weight.
Individual dose volume": Calculated from the most recently recorded scheduled bodyweight.
Control (Group 1): Vehicle at the same volume dose as treated groups.
Frequency: Females were treated from Day 6to Day 19(inclusive) after mating, once daily at approximately the same time each day.
Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations in the concentration range 1to 100 mg/mL were shown to the stable for 15 days following refrigerated storage (2-8ºC); following ambient storage (15-25ºC) formulations at 1 mg/mL were shown to be stable for 4hours and formulations at 100 mg/mL were stable for 24hours.

The samples were analyzed in accordance with the Envigo Analytical Procedure KR96BN-03R developed in study KR96BN and validated and reported in study FV31TP (GLP).The analytical method involved extraction in acetonitrile and dilution in acetonitrile/water (50/1 v/v) followed by gaschromatographic analysis withflame ionization detection(GC-FID). Sample concentrations were determined with reference to external standards prepared in the concentration range 10 µg/mlto 50µg/ml. Procedural recovery samples were prepared and analyzed concurrently with samples.

On Days 6 and 19 of the Preliminary phase and Days 6 and 19 of the Main phaseof the study, freshly prepared test formulations were sampled (1×10mL) by Pharmacy personnel and submitted for analysis. Duplicate aliquots (1 mL) of each formulationwere analyzed in accordance with the analytical procedure, and the remaining samples were retained for contingency.

The mean concentrations of N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)Tripropanamine in test formulations analyzed during the study were within ±8% of nominal which is within the applied limits +10/-15%, confirming the accuracy of formulation.
The percentage difference from the mean values were within ±2.0% confirming the precision of the analysis.
Details on mating procedure:
Male/female ratio: 1:1 with identified stock males.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation: When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females were treated daily by oral gavage from Day 6to Day 19(inclusive) after mating, once daily at approximately the same time each day.
Volume dose: 10mL/kg body weight.

Frequency of treatment:
Daily
Duration of test:
Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control Group (Vehicle - water)
Dose / conc.:
80 mg/kg bw/day
Dose / conc.:
240 mg/kg bw/day
Dose / conc.:
720 mg/kg bw/day
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Examinations

Maternal examinations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
- Pre-dose observation
- One to two hours after completion of dosing
- As late as possible in the working day
Ovaries and uterine content:
For females surviving to term, Uterus Gravid uterine weight (including cervix and ovaries).was recorded:

For all animals, each ovary/uterine horn was examined for numbers of:
- Corpora lutea.
- Implantation sites.
- Resorption sites (classified as early or late).
- Fetuses (live and dead).
Fetal examinations:
All viable fetuses and placentae were examined after dissection from \he uterus
Fetuses were individually weighed and identified within the litter using a coding system based on their position in the uterus. All fetuses were examined externally with any abnormalities recorded. The sex of each fetus was recorded.

50% of fetues in each litter were sexed internally and eviscerated.

Statistics:
For all adult parameters, the analyses were carried out using the individual animal as the basic experimental unit.
For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
- Body weight, using absolute weights and gains over appropriate study periods
- Gravid uterine weight and adjusted body weight
- Food consumption, over appropriate study periods
- C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
- Placental, litter and fetal weights

A sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio -percentage male, placental, litter and fetal weights:
- A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences.
- A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences.
Historical control data:
The Han Wistar (RccHan™ ;WIST) strain was used because of the historical control data available at this laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Administration of the test item at dose levels up to and including 720 mg/kg/day was well tolerated. There were no unscheduled deaths, no test item-related changes in general clinical condition and no signs observed in relation to dose administration at any dose level investigated.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment on overall mean body weight gain at any dose level investigated, however at 720 mg/kg/day mean body weight gain during Days 6-9 of gestation was 50% lower than Controls, with this difference attaining statistical significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following the start of treatment on Day 6 of gestation, group mean food intake for females given 720 mg/kg/day was statistically significantly lower than Control during Days 6-10 of gestation (79% of Controls). Thereafter, the mean food intake of females in this groupwas essentially similar to that of the Controls. Mean food consumption for females given80 or 240 mg/kg/day was unaffected.
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:
no effects observed
Description (incidence and severity):
The mean gravid uterine weight on Day 20 of gestation was essentially similar in all groups. When overall mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight gain was similar in all groups of females and no effect of treatment with the test item was inferred.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no maternal macroscopic abnormalities detected at scheduled termination. In one litter in the 240 mg/kg/day group (No. 43) all of the placenta were swollen, and in one litter in the 720 mg/kg/day group, a single placenta was considered to be enlarged.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
At 720 mg/kg/day some evidence of maternal toxicity was observed, manifest as a 50% reduction in mean body weight gain during Days 6-9 of gestation associated with a 21% reduction in mean food intake during Days 6-10of gestation. Thereafter, the body weight gain and mean food intake of females in this group was essentially similar to that of the Controls. Mean body weight gain and food consumption for females given 80 or 240 mg/kg/day was unaffected.

Gravid uterine weight and adjusted body weight gain were unaffected at all dose levels investigated, and there were no test item-related maternal macroscopic abnormalities detected at scheduled termination.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Three Control females (No’s. 16, 17 and 18) and one female given 240mg/kg/day (No. 51) had a total litter resorption. Therefore, results are based on 16, 20, 19 and 20 females with live young in Groups 1-4, respectively.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses at any dose level investigated.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses at any dose level investigated.
Description (incidence and severity):
There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses at any dose level investigated.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
At 720 mg/kg/day some evidence of maternal toxicity was observed, manifest as a 50% reduction in mean body weight gain during Days 6-9 of gestation associated with a 21% reduction in mean food intake during Days 6-10of gestation. Thereafter, the body weight gain and mean food intake of females in this group was essentially similar to that of the Controls. Mean body weight gain and food consumption for females given 80 or 240 mg/kg/day was unaffected.

Gravid uterine weight and adjusted body weight gain were unaffected at all dose levels investigated, and there were no test item-related maternal macroscopic abnormalities detected at scheduled termination.

There was no effect of maternal treatment on litter data, as assessed by the mean numbersof implantations, resorptions, live young, sex ratio and pre-and post-implantation losses. Placental, litter and fetal weights were similar in all groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
720 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on mean placental, total litter or fetal weights at any dose level investigated.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses at any dose level investigated.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses at any dose level investigated.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on mean placental, total litter or fetal weights at any dose level investigated.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor fetal abnormalities, skeletal variants and fresh macroscopic abnormalities showed no relationship to maternal treatment with the test item.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor fetal abnormalities, skeletal variants and fresh macroscopic abnormalities showed no relationship to maternal treatment with the test item.
Visceral malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor fetal abnormalities, skeletal variants and fresh macroscopic abnormalities showed no relationship to maternal treatment with the test item.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The incidence of major and minor fetal abnormalities, skeletal variants and fresh macroscopic abnormalities showed no relationship to maternal treatment with the test item.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
720 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Results tables attached below.

Applicant's summary and conclusion

Conclusions:
Treatment of pregnant female Han Wistar rats with N,N,N’,N’,N”,N”-Hexamethyl-1,3,5Triazine-1,3,5(2H,4H,6H)-Tripropanamineat dose levels up to and including 720 mg/kg/day showed evidence of mild maternal toxicity in the highest dose group (reduction in mean body weight gain during Days 6-9 of gestation with recovery).

Gravid uterine weight and adjusted body weight gain were unaffected at all dose levels investigated, and there were no test item-related maternal macroscopic abnormalities detected at scheduled termination.

There was no effect of maternal treatment on litter data, as assessed by the mean numbersof implantations, resorptions, live young, sex ratio and pre-and post-implantation losses. Placental, litter and fetal weights were similar in all groups.

The incidence of major and minor fetal abnormalities, skeletal variants and fresh macroscopic abnormalities showed no relationship to maternal treatment

Based on the results obtained in this study, it was concluded that the high dose level of 720 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.
Executive summary:

In a GLP-compliant OECD 414 guideline study on Pre-Natal Developmental Toxicity (PNDT), three groups of 20 female Han-Wister rats received N,N,N’,N’,N”,N”-Hexamethyl-1,3,5-Triazine1,3,5(2H,4H,6H)-Tripropanamineat doses of 80, 240 or 720mg/kg/day by oral gavage administration at a volume dose of 10 mL/kg body weight, from Day 6 to19after mating, inclusive. A control group received the vehicle, purified water at the same volume dose as treated groups.  

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

At 720 mg/kg/day some evidence of maternal toxicity was observed, manifest as a 50% reduction in mean body weight gain during Days 6-9 of gestation associated with a 21% reduction in mean food intake during Days 6-10of gestation.  Thereafter, the body weight gain and mean food intake of females in this group was essentially similar to that of the Controls.  Mean body weight gain and food consumption for females given 80 or 240 mg/kg/day was unaffected.

Gravid uterine weight and adjusted body weight gain were unaffected at all dose levels investigated, and there were no test item-related maternal macroscopic abnormalities detected at scheduled termination.  

There was no effect of maternal treatment on litter data, as assessed by the mean numbersof implantations, resorptions, live young, sex ratio and pre-and post-implantation losses.  Placental, litter and fetal weights were similar in all groups.  

The incidence of major and minor fetal abnormalities, skeletal variants and fresh macroscopic abnormalities showed no relationship to maternal treatment with the test item.

It was concluded that the high dose level of 720 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.