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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
April-November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
No data are available on the developmental toxicity/teratogenicity of the test substance (TRIS AMINO). The justification for read-across has been attached in section 13 of this IUCLID. A GLP-compliant prenatal development study in the rat via the oral route following OECD 414 has been conducted, using the analogue substance 2-amino-1,3-propanediol (APD) as test substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Serinol (APD-1,3)

Specific details on test material used for the study:
Name: 2-Aminopropane-1,3-diol
Other name: Serinol, APD (Aminopropanediol)
Molecular formula: C3H9NO2
CAS number: 534-03-2
Batch number: F100G6RR10
Appearance: White solid
Purity: 100%
Manufacturer: ANGUS Chemical Company
Manufacture date: 14 January 2016
Expiry date: 27 June 2017
pH: 11

Storage conditions: Room temperature, under inert gas (15-25 Celsius, below 70 RH%)
Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials will be applied to assure personnel health and safety. Dangerous to the environment.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Species and strain: Hannover Wistar rats (CRLHan)
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
Housing condition: Standard laboratory conditions; individual housing
Target age of animals: Young adult female rats, nulliparous and non-pregnant, at least approximately 11 weeks old
Target body weight: Will not exceed ± 20% of the mean weight at onset of treatment
Acclimatisation period: At least 5 days

Animal health: Only healthy animals will be used for the test, as certified by the staff veterinarian.
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany), or similar laboratory bedding suitable for the purposes of the study.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents will be housed individually. Nest building material (ARBOCEL natural crinklets produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, Rosenberg, D-73494 Germany), or similar will be also added to the cages – the certificate of analysis will be included in the report) will be added to the cages.

The temperature and relative humidity values were measured twice daily during the acclimatisation period and during the experiment.

The animals will be provided with ssniff® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water as for human consumption, ad libitum. The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier will provide an analytical certificate for the batch used.

The quality control analysis of the water is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis will be included in the raw data, and will be archived at CiToxLAB Hungary Ltd.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd. During the formulation process, the pH of each formulation will be adjusted using 10% HCl solution to achieve a pH<10, this fact will be documented in the raw data and reported.ormulations were prepared fresh prior to administration to animals or at appropriate frequency to allow their use according to stability assessments. Stability of the 2-aminopropane-1,3-diol in the vehicle (distilled water) was assessed in the
conditions employed on the study during the validation study.

The analysis of test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples will be taken from the test item formulations, each analysed at least two times during the study (approximately during the first and last week of treatment). Similarly, one sample will be taken in duplicate on each occasion from the middle of the control (vehicle) formulation for concentration measurement.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis of test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. The contentd of 2-aminopropane-1,3-diol and homogeneity of the dosing formulations was determined on two analytical occasions during the test. Analytical samples were taken from each test concentration to determine concentration and homogeneity and from the vehicle control. The samples were derivatized with trifluoroacetic acid diluted into the calibrated range with water then analysed by GC method.
.
Details on mating procedure:
The oestrus cycle of female animals was examined shortly before start of pairing. After acclimatisation the females, according to their oestrus cycle, were paired with males for approximately 2 hours (1 male: 1-3 females) in the morning, until at least 24 sperm-positive females/group are obtained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm-positive females were separated and caged individually.

The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day.
Duration of treatment / exposure:
The control or test item dose formulations was be administered to mated, spermpositive assumed pregnant female rats daily by oral gavage on a 7 days/week basis from Gestation Day (GD) 6 to GD19.
Frequency of treatment:
Daily from GD 4 till GD 19.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Actual dose: 92 mg/kg bw
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Acutal dose: 294 mg/kg bw
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Actual dose: 940 mg/kg
No. of animals per sex per dose:
24 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE
The dose levels were set based on available data, including the results of a Dose Range Finding (DRF) study by oral gavage in rats and another DRF study in pregnant rats, with the aim of inducing toxic effects but no death or suffering at the highest dose (1000 mg/kg bw/day) and a NOAEL at the mid dose (300 mg/kg bw/day) or at the lowest dose (100 mg/kg/bw/day). In the pregnant DRF study, no signs of maternal toxicity, embryotoxicity or foetotoxicity were observed in any test item treated dose groups (up to and including 1000 mg/kg bw/day). Based on these results, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study. The oral route is a potential route of human exposure to the test item and is considered suitable to provide the exposure required for this developmental toxicology study. A constant volume of 10 mL/kg bw was administered to all dose groups, including the controls. The individual volume of the treatment wasl be based on the most recent individual body weight of the animals.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

CLINICAL OBSERVATIONS: Yes
Clinical observations were made twice daily (at the beginning and end of each working day, or before treatment and when the peak of the clinical observations, if any, is observed after treatment). Only one clinical observation was made in the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, clinical observations were made only once on necropsy days. Detailed clinical observations were made on all animals at the onset of treatment (GD6) then at least weekly and on the necropsy days. Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated included, but were not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also be recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. On GD13 and/or 14 the sperm-positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

BODY WEIGHT: Yes
Body weight of each animal was recorded on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
Food was measured on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was calculated for each interval, including GD 6-20 and GD 0-20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. Sodium pentobarbital administered by intraperitoneal injection, followed by exsanguination was used for euthanasia (pentobarbital sodium for injection.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes; stomach of all animals (which will be examined thoroughly) and all the gross findings will be retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, if any abnormalities noted) for possible future evaluation. The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and held in front of a light source to clearly identify the implantation sites. Uteri that appear non-gravid were further examined to confirm the non-pregnant status (i.e. by ammonium sulphide staining or a suitable alternative method). The corrected body weight was calculated (body weight on GD20 minus weight of the gravid uterus). The number of corpora lutea in each ovary and of implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted; the number and percent of pre- and postimplantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
Fetal examinations:
Each live foetus were weighed individually and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the anogenital distance. Thereafter, the foetuses were individually identified; approximately half of each litter were subjected to visceral examination, and the other half will be processed for skeletal examination. For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened, and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomya mixture: then after fixation, the body will be micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed; the cadavers were fixed in Alcian-blue -acetic acid-ethanol mixture. After fixation in isopropanol, the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope. All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded.
Statistics:
Data were collected using the software PROVANTIS v.9, or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the software Microsoft Office Word and/or Excel, as appropriate. Data were collected to provide information on parameters including:

MATERNAL DATA:
- Number of animals at test start, no. of animals surviving, no. of pregnant animals, no. of animals with total intrauterine mortality
- Clinical signs (by gestation day)
- Mortality (by gestation day), if any
- Body weight and body weight gain: mean ± S.D.
- Corrected body weight on GD20 (body weight on GD20 minus gravid uterine weight) and corrected body weight gain (body weight gain of GD0-20 minus
gravid uterine weight): mean ± S.D.
- Net body weight change (body weight gain of GD6-20 minus gravid uterine weight): mean ± S.D.
- Gravid uterine weight: mean ± S.D.
- Food consumption: mean ± S.D.
- Gross pathology findings,

CAESAREAN SECTION AND NECROPSY DATA:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percentage of live foetuses: mean ± S.D.
- Number and percentage of intrauterine mortality: mean ± S.D. Classified according to time of death: preimplantation loss, postimplantation mortality, early and late embryonic as well as foetal death.
Indices:
- Preimplantation loss: %, group mean: Number of corpora lutea-Number of implantations x 100 / Number of corpora lutea
- Postimplantation loss: %, group mean: Number of implantations-Number of live foetuses x 100 / Number of implantations

Foetal Data:
- Sex distribution: %, group mean: Number of male (female) foetuses x 100 / Number of foetuses
- Foetal body weight (accuracy 0.01 g): mean ± S.D.
- External abnormalities/litter: %, group mean: Number of foetuses with abnormality x 100 / Number of foetuses
- Visceral abnormalities/litter: %, group mean: Number of foetuses with abnormality x 100 / Number of foetuses
- Skeletal abnormalities/litter: %, group mean: Number of foetuses with abnormality x 100 / Number of foetuses

The statistical evaluation of data was performed with the program package SPSS PC+4.0 (SPSS Hungary Ltd., Budapest, Hungary) or SAS v9.2 (when using
Provantis).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related adverse effects or systemic clinical signs were noted in the treated animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no unscheduled mortality during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item related effect on body weight was observed in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day) when compared to control. No toxicologically significant changes were observed in the mean body weights of the Low, Mid or High dose groups when compared to control. Similarly, no statistically significant or biologically relevant differences were seen in the body weight gain, corrected body gain or net body weight gain values during the treatment period (GD 6-20) or entire study (GD 0-20) compared to the control value
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No biologically relevant changes were observed in the mean daily food consumption of dams in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively) compared to the control value. The food consumption of the Low, Mid and High dose groups was comparable with the Control group, the sight differences in the daily mean food consumption of the High dose group during the different periods were not larger than 8.0% when compared to the control. There was no statistical significance and clear dose response, furthermore based on the absolute values those differences were considered as being non-test item related background variations.
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:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups. There was no statistically significant difference in the preimplantation loss of the test item treated groups when compared to the control. The early and the late embryonic loss did not differ significantly from the control in the test item treated groups. There was no statistically significant difference in the post-implantation loss between the test item treated and control groups.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in foetal death in any test item treated groups compared to the control
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:
No test item related observations were recorded for any evaluated animals in the study.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: no effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The weight of foetuses per litter in the Low and Mid dose groups (100 and 300 mg/kg bw/day, respectively) did not differ significantly from the control mean value. In the High dose group (1000 mg/kg bw/day), mean foetal weight per litter was significantly (p<0.05) higher than control value, but based on the biological relevance this fact was considered as a non-adverse test item related effect.

Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of viable foetuses was comparable with the control mean in all test item treated groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The weight of foetuses per litter in the Low and Mid dose groups (100 and 300 mg/kg bw/day, respectively) did not differ significantly from the control mean value. In the High dose group (1000 mg/kg bw/day), mean foetal weight per litter was significantly (p<0.05) higher than control value, but based on the biological relevance this fact was considered as a non-adverse test item related effect. The total number of retarded foetuses (runts) as well as the number of affected litters was comparable to control in all test item treated groups indicating no test item effect on this parameter.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no recorded external variations in this study. One malformation was recorded in 1 out 228 foetuses in the high dose group.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Most of the skeletal findings corresponded with the current study control / HC data, or were incidental findings without dose response. However, in case of some skeletal variations (see further in this section), test item related increased incidence was observed in the High and Mid dose group, but no effect was noted in the Low dose group. The total number of foetuses with skeletal malformations did not differ from the control in a biologically relevant way (there was no clear dose response and the litter based incidence can not be clearly differentiated from an incidental frequency). The total number of foetuses with skeletal variations was significantly (p<0.01) higher in the High (1000 mg/kg bw/day) dose group than in the Control group, slightly increased number without statistical significance was detected in the Mid dose group (300 mg/kb bw/day), while no similar difference was noted in the Low dose group (100 mg/kg bw/day). Consequently, the total number of intact foetuses was lower than control in the High dose group. These findings indicated a slight test item effect in the High dose group.


Visceral malformations:
no effects observed
Description (incidence and severity):
All of the visceral findings are consistent in general nature and incidence with the study concurrent control data or the existing historical control data or were incidental findings without dose response, therefore considered as not related to the test item treatment. Based on the visceral findings, the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).

Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Most of the findings correspond to the study control data or have isolated occurrence that were considered incidental ascribed to individual variability. In the case of a few skeletal variations, an apparent increased incidence of changes in absolute numbers and percentile litter mean were observed with statistical significance in the high dose when compared to control (skull, hyoid body, unossified; ossified sternebra (3 or less); vertebra, unossified; carpal, ossified ≤ 2.5). These variations are all associated with delayed ossification at skeletal development. Based on these results the test item did not cause a higher incidence of malformations, but some skeletal variations in the high dose group, related to delayed ossification, appeared to be related to treatment.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: forelimb
skeletal: sternum
skeletal: vertebra

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)

Applicant's summary and conclusion

Conclusions:
In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses other than skeletal variations related to delayed ossification during skeletal development. The No-Observed-Adverse-Effect Levels (NOAEL) for maternal toxoicity, embryotoxicity, foetotoxicity, foetal malformations and skeletal development were determined to be 1000 mg/kg bw/day. The No-Observed-Effect Level (NOEL for skeletal development was 100 mg/kg bw/day
Executive summary:

The test item 2-aminopropane-1,3-diol (APD) was administered daily by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19). There was no test item effect on maternal body weight / body weight gain, corrected body weight /body weight gain and on food intake in any test item groups up to and including 1000 mg/kg bw/day. There were no toxicologically significant differences, or test item related-changes in the evaluated intrauterine parameters (including number of corpora lutea and implantation site, pre- and post-implantation loss, number of foetal death and total intrauterine mortality) up to and including 1000 mg/kg bw/day. No remarkable test item-related internal or external observations were recorded for any pregnant animals during necropsy. No remarkable abnormalities were observed on the placentas in any examined groups. No toxicologically relevant adverse effect of the test item was observed on the foetal parameters (number of viable foetuses, sex distribution of foetuses, mean foetal weight per litter, number of foetuses with retarded body weight) up to and including 1000 mg/kg bw/day. There was no biologically relevant and/or statistically significant increase in external, visceral and/or skeletal malformations in any test item treated groups when compared to control. Some skeletal variations ascribed to slightly retarded ossification (unossified hyoid body on the skull, ossified sternebra (3 or less); unossified vertebra and carpal, ossified ≤ 2.5) showed higher incidence (mostly with statistical significance) in the high dose and mid group. These variations are all associated with delayed ossification at skeletal development, commonly related to retarded foetal development. No signs of maternal toxicity were detected during the study based on clinical signs and bodyweight; however, in the 90-day rat repeated dose toxicity study with this test item, systemic toxicity was observed, in the absence of body weight or clinical signs. The foetal skeletal findings were considered to reflect a non-adverse, slight retardation of ossification.

 

In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses other than skeletal variations related to delayed ossification during skeletal development. The No-Observed-Adverse-Effect Levels (NOAEL) for maternal toxoicity, embryotoxicity, foetotoxicity, foetal malformations and skeletal development were determined to be 1000 mg/kg bw/day. The No-Observed-Effect Level (NOEL for skeletal development was 100 mg/kg bw/day