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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:
16 September 2008 to 08 April 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
(2,3-dihydroxypropyl)trimethylammonium chloride
EC Number:
251-783-2
EC Name:
(2,3-dihydroxypropyl)trimethylammonium chloride
Cas Number:
34004-36-9
Molecular formula:
C6H16NO2.Cl
IUPAC Name:
1-Propanaminium, 2,3-dihydroxy-N,N,N-trimethyl-, chloride (1:1)
Test material form:
solid - liquid: aqueous solution
Details on test material:
- Appearance: Clear colourless liquid
- Storage condition of test material: Stored at room temperature (10-30 °C) in the dark
- Stability: Stable under storage conditions

Test animals

Species:
rat
Strain:
other: Rat, HanRcc: WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland.
- Age at study initiation (Day 0 Post Coitum): 11 weeks
- Weight at study initiation (Day 0 Post Coitum): 180-224 g
- Housing: Animals were housed individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
- Diet: Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70%
- Air changes: Air-conditioned with 10 - 15 air changes per hour
- Photoperiod: 12-hour fluorescent light / 12-hour dark cycle with music during the light period

IN-LIFE DATES: 16 September 2008 to 08 April 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q-Water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly and to 100% to compensate for purity. Therefore, a correction factor of 1.84 was used. The appropriate volume of test substance was accurately measured by volume into a glass beaker and the vehicle added (volume:volume). The mixture was prepared using a magnetic stirrer.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer for at least 10 minutes before and during the dosing procedure.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored in the refrigerator (2 - 8 °C) in brown glass beakers. The dose formulations were stable up to one week according to information provided by the Sponsor. The dose formulations were divided into daily aliquots. Aliquots were allowed to equilibrate to room temperature before dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day (day 1 post coitum) and the last treatment day (day 20 post coitum) samples of 2 mL were taken and weighed from the middle of the dose formulations for the control group and each treatment group prior to dosing, for analysis of concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the principal investigator and stored there at -20 ± 5 °C until analysis.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation:
- Verification of same strain and source of both sexes: Yes; Male rats of the same source and strain were used only for mating. These male rats are in the possession of Harlan Laboratories and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.
- Proof of pregnancy: copulation plug / sperm in vaginal smear referred to as day 0 post coitum
Duration of treatment / exposure:
Day 1 - 20 post coitum
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
22 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar rats, RCC Study Number C06236, using dose levels of 100, 300 and 1000 mg/kg bw/day.
- Rationale for animal assignment: Computer-generated random algorithm

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).

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

FOOD CONSUMPTION: Yes
- Recorded at intervals of: days 0-1, 1-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- At the scheduled necropsy on day 21 post coitum, females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.
- All females sacrificed were subjected to macroscopic examination with emphasis on the uterus and its contents. Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
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
Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter was fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in plastic vials.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
Fetuses with abnormalities were photographed.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Historical control data:
The data was compared with historical control data of the laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No signs of discomfort or clinical symptoms from the treatment with the test item were observed.
- In group 2, female no. 25 had a hairless region on both forelegs from day 17 post coitum until the end of the treatment period.
Mortality:
no mortality observed
Description (incidence):
All females survived until the end of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Mean body weight and body weight gain (+65.2%, +66.0%, +65.2% and +65.7% in order of ascending dose level) were not affected by treatment with the test item in any group.
- Mean corrected body weight gain (corrected for the weight of the gravid uterus) was similar in all dose groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption was considered to be not affected by treatment with the test item in any group (+2.2%, ±0% and -3.1% in groups 2, 3 and 4, respectively, compared to the control group).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
During macroscopic examination, no abnormal findings were noted.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Incidentally, the pre-implantation loss was statistically significantly lower in group 3 compared to the control group, resulting in a statistically significantly higher mean number of implantation sites.
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There was no test item- and/or dose-related effect on the reproduction parameters in any dose group. The mean number of fetuses per dam was 11.7 12.4, 12.9 and 12.0 in order of ascending dose level.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Remarks:
maternal toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
mortality
pre and post implantation loss

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Mean fetal weights were not affected by treatment with the test item.
- Mean fetal weights were 4.9 g, 4.9 g, 4.8 g and 5.1 g in order of ascending dose level when calculated on a litter basis for combined data of male and female fetuses.
- The statistically significantly higher mean fetal weights noted in group 4 were borderline to the range of the historical control data (4.8 - 5.0 g) and were therefore considered to be incidental.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- No test item-related effects on the sex ratio of the fetuses were noted in any group.
- The proportion of male fetuses was 49.8%, 49.3%, 46.7% and 57.4% in order of ascending dose level.
External malformations:
no effects observed
Description (incidence and severity):
- No test item-related findings were noted during external examination of the fetuses in any group.
- In group 2, two fetuses from two litters had a hematoma on the trunk and hindlimbs or on the head.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During skeletal examination of the fetuses, findings were noted in:
21% examined fetuses (in 81% litters) in group 1
11% examined fetuses (in 50% litters) in group 2
12% examined fetuses (in 62% litters) in group 3
14% examined fetuses (in 41% litters) in group 4
- No test item-related findings were noted.
- Bone abnormalities were found in 2 fetuses (2 litters) in group 2. Fetus no. 115 had a cervical vertebral body fused to thoracic vertebral body and fetus no. 364 had fused ribs and one thoracic vertebral body was absent. One cartilage abnormality was found in one fetus in group 4, which had a split cervical vertebral body. These are common findings in the control population, and their low incidence did not indicate an effect of treatment.
- The type and frequencies of commonly noted variations were similar in nature for the groups receiving the test item and the control group and did not indicate any dose-dependency. They were therefore considered not to be test item-related effects.

Bone Examination - Ossification Stage / Supernumerary Ribs
Reduced Alizarin Stain Density
- Routine skeletal examinations revealed that fetuses processed in two particular containers had poor alizarin stain uptake. The principle issue appeared to reduced stain density (pale stain colour) but, in some structures, stained area was also reduced. The alizarin staining was either poor throughout the skeleton or it was restricted to the extremities (eg paw structures, caudal vertebrae, and in some cases, the distal extremities of the ribs and the lower sternebrae). The cause of the poor stain uptake in these two containers was considered to be an adverse event during skeletal processing.
- The detection of structural changes was unaffected by the processing issue and only findings related to stage of ossification were concerned. Therefore, although fetuses in the affected containers were excluded from ossification summaries, all fetuses were included in all other summaries of skeletal examination data (this includes structural abnormality and variation values for both bone and cartilage).
- A total of 137, of the 504 fetuses allocated to skeletal processing, was affected by the poor stain uptake. The intergroup distribution of affected fetuses was 17 (3 litters), 36 (6 litters), 42 (7 litters) and 42 (7 litters) in groups 1, 2, 3 and 4. A total of 100 (18 litters), 95 (16 litters), 87 (14 litters) and 85 (15 litters) in groups 1, 2, 3 and 4 were evaluated for stage of ossification. This was considered a sufficient number to not affect the integrity of the study.

Stage of Ossification Findings/ Supernumerary Ribs
- There were no test item-related effects on the ossification stage or the number of supernumerary ribs.
- There were significantly fewer fetuses at 1000 mg/kg bw/day with non ossified talus compared with the control group. Since there was no significant difference in the number of litters affected this difference was considered not to represent an effect of treatment with the test item. There were significantly fewer fetuses (but not litters) at 100 mg/kg bw/day with supernumerary ribs. Since there were no differences at higher doses this difference was also considered incidental.

Cartilage Examination - Additional Variations
- During the examinations no test item-related findings were noted in the common cartilage variations.
- In groups 2 and 4, a statistically significantly lower incidence of interrupted costal cartilage 10 was noted, when calculated on a litter basis. Additionally in group 4, a lower incidence of branched xiphoid cartilage was noted. Due to the absence of a dose dependency these findings were considered to be incidental.
- The alcian blue staining was unaffected in all fetuses, which had a poor alizarin stain uptake and all cartilage structures could be routinely evaluated.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During visceral examination of the fetuses, findings were noted in:
37% examined fetuses (in 90% litters) in group 1
38% examined fetuses (in 100% litters) in group 2
40% examined fetuses (in 90% litters) in group 3
34% examined fetuses (in 91% litters) in group 4
- No test item-related findings were noted.
- Abnormalities were found in one fetus each in groups 1 and 3, in 3 fetuses (3 litters) in group 2 and in 2 fetuses (2 litters) in group 4. In group 1, one fetus had an internal hydrocephaly and in group 3 disorganized eye tissues was noted for one fetus. In group 2, for fetus no. 93 a misshapen and malpositioned pituitary and a dilated ascending aorta were noted, fetus no. 412 had a small pituitary and fetus no.1002 a severely dilated renal pelvis. In group 4, for fetus no. 264 a microphthalmia and for fetus no. 556 a severely dilated renal pelvis were noted. These are common findings in the control population, and their low incidence did not indicate an effect of treatment.
- The type and frequencies of commonly noted variations were similar in nature for the groups receiving the test item and the control group and did not indicate any dose-dependency. They were therefore considered not to be test item-related effects.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Remarks:
fetal toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOEL for maternal and fetal toxicity was considered to be 1000 mg/kg bw/day. Under the conditions described for this study, test substance did not reveal teratogenic potential up to and including 1000 mg/kg bw/day.
Executive summary:

In a Prenatal developmental toxicity study performed in accordance with OECD test guideline No. 414 and in compliance with GLP, test substance was administered daily by oral gavage to SPF-bred Wistar rats (22 mated females per group)at dose levels of 100, 300 and 1000 mg/kg bw/day from day 1 post coitum (after mating) to day 20 post coitum (the day prior to Caesarean section). A control group was treated similarly with the vehicle, Milli-Q-Water. All females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. During the study, clinical condition, body weight, food consumption, macropathology investigations were undertaken (maternal toxicity data). Fetal data: External examination, sex ratios, body weights, visceral examination, skeletal and cartilage examination were performed.

 

Maternal Data

All dams survived until the scheduled necropsy. No clinical symptoms related to treatment with the test item were noted during the study. Mean food consumption, mean body weight and corrected body weight gain (corrected for the gravid uterus weight) were not affected by treatment with the test item in any dose group. Post-implantation losses and the mean number of fetuses per dam were not affected by treatment with the test item at any dose level. No macroscopical findings were noted during necropsy of the dams.

 

Fetal Data

During the external examination of the fetuses, no test item-related abnormal findings were noted. No test item-related effects on fetal sex ratios were noted in any dose group. No test item-related effects on fetal body weights were noted. No test item-related abnormalities were noted during the visceral examination of fetuses. No abnormalities, which were considered to be test item-related, were noted during examination of fetal skeletons and cartilages.

 

Based on the results of this study, the NOEL for maternal and fetal toxicity was considered to be 1000 mg/kg bw/day. Under the conditions described for this study, test substance did not reveal teratogenic potential up to and including 1000 mg/kg bw/day.