Sodium 3-(allyloxy)-2-hydroxypropanesulphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17. Februar 2016 - 120. Februar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-(allyloxy)-2-hydroxypropanesulphonate

EC Number:

258-004-5

EC Name:

Sodium 3-(allyloxy)-2-hydroxypropanesulphonate

Cas Number:

52556-42-0

Molecular formula:

C6H12O5S.Na

IUPAC Name:

sodium 2-hydroxy-3-(prop-2-en-1-yloxy)propane-1-sulfonate

additive 1

Reference substance name:

Sodium hydroxide

EC Number:

215-185-5

EC Name:

Sodium hydroxide

Cas Number:

1310-73-2

Molecular formula:

HNaO

IUPAC Name:

sodium hydroxide

impurity 1

Reference substance name:

3-(allyloxy)propane-1,2-diol

EC Number:

204-620-4

EC Name:

3-(allyloxy)propane-1,2-diol

Cas Number:

123-34-2

Molecular formula:

C6H12O3

IUPAC Name:

3-(allyloxy)propane-1,2-diol

impurity 2

Reference substance name:

disodium 2-hydroxy-3-(3-sulfonatopropoxy)propane-1-sulfonate

Molecular formula:

C6H12Na2O8S2

IUPAC Name:

disodium 2-hydroxy-3-(3-sulfonatopropoxy)propane-1-sulfonate

Test material form:

solid - liquid: aqueous solution

Details on test material:

light yellow liquid

Specific details on test material used for the study:

The test item was administered at appropriate concentrations, prepared with the vehicle (dilution with distilled water).
The pH value of the formulations was checked and adjusted to pH 6.0-7.0 using 85% ortho-phosphoric acid. Preparation of the test item formulations was made using a magnetic stirrer with a frequency of one to three days. The formulations were stored in a refrigerator (at 5±3 oC). According to the results of the stability measurements in course of the Method Validation the stability of the test item in water was at least 1 day at room temperature and 3 days at 5±3 oC at the concentration levels of 2.5 mg/mL and 550 mg/mL.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Housing: pre-mating period: 2-3 females per cage
2 males per cage

during mating hours: 1 male with 1- 3 females;
during gestation: 2-3 sperm positive females per cage

Environmental Conditions

Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 -23 °C
Relative humidity: 30 - 45 %
Ventilation:above 10 air exchanges/hour by central air-conditioning system.

Environmental conditions were maintained by an air-condition system. Temperature and relative humidity were verified and recorded daily during the study

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the course of this study an HPLC-UV method has been validated for the analysis of Test Item in water.
The analytical method was successfully validated, in accordance with the parameters stipulated in the study plan. The procedure was found to be suitable
for the analysis, the measured parameters are summarised in the following Table.


Selectivity no interfering peak was observed
Repeatability (7 replicates) CV% ≤ 0.9 %
Linear range 50 - 1500 µg/mL
Limit of Quantification 50 µg/mL
Recovery of Test Item from Water 94% at 2.5 mg/mL concentration level (RSD ≤ 0.4)
100% at 550 mg/mL concentration level (RSD ≤ 0.6)
Stability of the test item in Water at room temperature after 1 day 101 % (ca. 2.5 mg/mL)
101 % (ca. 550 mg/mL)
Stability of the test item in Water at 5 ± 3 °C after 3 days 101 % (ca. 2.5 mg/mL)
94 % (ca. 550 mg/mL)
Stock solution stability at least 3 days at 5  3 °C
Stability in the autosampler at least 21 hours

Details on mating procedure:

The females were paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females / group achieves twenty two. Vaginal smears were prepared from each female, stained with 1 % aqueous methylene blue solution and examined for presence of sperm and for estrus cycle. The day of mating is regarded as day 0 of pregnancy (vaginal plug and/or sperm in the vaginal smear). Sperm positive females were separated and caged in groups of 1 to 3 animals, however individual caging was avoided if possible.

Duration of treatment / exposure:

From day 6 up to and including day 19 post coitum

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

The dose levels refer to the Sodium 3-allyoxy-2-hydroxy-1-propanesulfonate quantity in the dosing solutions calculated with 38.2 wt% in mixture.

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

The dose levels refer to the Sodium 3-allyoxy-2-hydroxy-1-propanesulfonate quantity in the dosing solutions calculated with 38.2 wt% in mixture.

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

The dose levels refer to the Sodium 3-allyoxy-2-hydroxy-1-propanesulfonate quantity in the dosing solutions calculated with 38.2 wt% in mixture.

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS
General clinical observations of the sperm positive females was made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. Individual observation included the check of behavior and general condition.

BODY WEIGHT
The body weight of the male animals was not measured.

The body weight of the female rats was measured at least once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 6, 9, 12, 15, 18 and 20 (accuracy of 1 g).
Corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption was measured between gestation days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 by re-weighing the non-consumed diet.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No

Fetal examinations:

The number of litters with malformed fetuses was two in the 100 and three in the 1000 mg/kg bw/day dose group. There were no malformations found in the control and 300 mg/kg bw/day dose group.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Alopecia of one female was observed in the 300 mg/kg bw/day group which was not attributed to the treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight of the females was similar in all groups on each measurement day.
There were no treatment related differences in the body weight gain of the animals. Between gestation days 6 and 9 there was a statistically significant (p<0.05) increase of body weight gain indicated in the 300 mg/kg bw/day group which was judged to be without a dose response. There were no significant differences in the mean corrected body weight and body weight gain of the animals in the different groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption of the dams in the 1000 mg/kg bw/day group was slightly lower between days 6 and 9 as well as between days 9 and 12. Though a statistical significance (p<0.01 and p<0.05 respectively) considering the slight manner (-6% in both periods), these differences were considered as non-adverse. Statistically significant decrease (p<0.01) was also indicated in the pre-treatment period in the 100 mg/kg bw/day group group without a biological relevance.

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):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight of the females was similar in all groups on each measurement day.
There were no treatment related differences in the body weight gain of the animals. Between gestation days 6 and 9 there was a statistically significant (p<0.05) increase of body weight gain indicated in the 300 mg/kg bw/day group which was judged to be without a dose response. There were no significant differences in the mean corrected body weight and body weight gain of the animals in the different groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no macroscopic changes recorded for the females in the experimental groups.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no macroscopic changes recorded for the females in the experimental groups.

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Control group: 1 total postimplantation loss
300mg/kg bw/day: total postimplantation loss

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

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

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

dead fetuses

early or late resorptions

effects on pregnancy duration

maternal abnormalities

number of abortions

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

not specified

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no differences indicated attributed to the test item.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no treatment related differences in the mean body weight of the fetuses (also not if combined by sex) as well as in the placental- and relative placental weight. The mean body weight of female fetuses was statistically significantly (p<0.05) higher in the 100 mg/kg bw/day group, however the difference was only 0.1 g and no dose response was indicated.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Body weight retardation (limit: below 2.85 g for males and below 2.83 g for females) was evaluated as an external variation. There were no significant differences in the fetal- and litter incidences.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At external examination two fetuses were found with short tail and one of them with hypoplastic pollex (not proved at skeletal examination) in the 100 mg/kg bw/day dose group. Both of these fetuses had multiple malformed vertebrae and in addition one of them had fused ribs. In this group a third fetus had also fused ribs and multiple malformations of the thoracic vertebrae.
In the high dose group two fetuses were found with bent scapula or/and ulna or/and slightly shorter femur. A third fetus in the high dose group had a bipartite thoracic vertebra with dumb-bell shaped cartilage. All of the malformations found in the low and high dose group occur sporadically also in control rat fetuses according to the Background pregnancy and fetal data of Toxi-Coop Zrt. (Appendix XXIV/A) and occurred with low incidence or without a dose response and were considered to be without a test item relationship in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were three malformations found each in the high and low dose group and none in the mid dose.
In the high dose group one fetus was found with bent scapula (bilateral), bent ulna (unilateral) and slightly shorter femur (unilateral). Another fetus had bent scapula (bilateral). The third fetus had a bipartite thoracic vertebra with dumb-bell shaped cartilage. All of the malformations found in the high dose group occur sporadically also in control rat fetuses according to the Background pregnancy and fetal data of Toxi-Coop Zrt. (Appendix XXIV/A) and were considered to be without a test item relationship in this study.
The two fetuses found with short tail at external examination in the 100 mg/kg bw/day group in a common litter were proved to have multiple malformed vertebrae and one of these fetuses had also fused ribs.
One Fetus had fused arches and bipartite cartilage of cervical vertebrae, fused, hemicentric, in cartilage bipartite thoracic vertebrae, hemicentric, misshapen lumbar cetnra, absence of a part of caudal vertebrae and in addition variations such as asymmetric, dumb-bell shaped thoracic centra and slightly dumb-bell shaped cartilage. The neck rib was fused with the first rib bilateral in this fetus.In the other fetus of the same litter fusion was observed of the exoccipital with the first cervical vertebra as well as bipartite cartilage or fusion of other cervical vertebrae; misshapen cartilage of thoracic centra; hemicentric thoracic and lumbar centra; fused cartilage of thoracic and lumbar vertebrae; displaced and misshapen lumbar centra; dumb-bell shaped cartilage of sacral centrum; abnormal flexion of caudal part of vertebral column; fusion of first caudal vertebrae with Sacral IV and absence of a part of caudal vertebrae. In addition vertebral variations such as dumb-bell shaped (including slightly dumb-bell shaped cartilage) and asymmetric bipartite or unossified thoracic centra were recorded for this fetus.
The third fetus in the 100 mg/kg bw/day group had fused ribs and misaligned, bipartite (including cartilage) thoracic centra; a hemicentric and displaced centrum of Th XI (the present half of centrum (left) was common with the right side of Th XII centrum).
The detailed description of the individual observations are included in Appendix XXI.
Considering the lack of dose response in case of multiple malformed vertebrae (with or without fused ribs) this malformations were not considered to be a consequence of the treatment. Moreover according to the background data of Toxi-Coop Zrt. (Appendix XXIV/A, B) multiple malformed vertebrae may occur incidentally in rat fetuses.

Variations:
Skeletal findings such as retardation of the skull, incomplete or irregular ossification of the skull bones, unossified hyoid bone, less than 3 ossified or bipartite sternebra, wavy ribs, a presence of a neck rib, dumb-bell shaped, bipartite and/or asymmetric vertebrae, slightly dumb-bell shaped cartilage of thoracic centra, unossified thoracic centra or lumbar arches, asymmetric pelvic articulation of sacral/lumbar arches, unossified pubic or ischii as well as asymmetric ossification of metacarpal/metatarsal or if less than 3/3.5 ossified were classified as variations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly dilated lateral brain ventricles or slightly dilated perimeningea space as well as pinched liver lobe were found only in the control group. Slightly dilated third brain ventricle was recorded for one fetus in the 100 mg/kg bw/day group. Hydroureter (or convoluted ureter in one fetus) as well as hydroureter with dilated renal pelvis and slightly malpositioned kidney was found with a low incidence and without a dose response. In the high dose group only hydroureter was found.

Other effects:

no effects observed

Effect levels (fetuses)

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:

external malformations

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Treatment related:

no

Relation to maternal toxicity:

not specified

Dose response relationship:

no

Applicant's summary and conclusion

Conclusions:

Based upon these data, treatment of pregnant Hsd. Han: WIST Rats from gestational day 6 to 19 by oral administration of Sodium 3-allyloxy-2-hydroxy-1-propanesulfonate (HAPS), caused no mortality, no clinical signs and necropsy alterations and no adverse effects on the body weight as well as food consumption of the maternal animals. The treatment of the dams did not increase the pre- and post-implantation loss and had no influence on the mean number of viable fetuses and their sex distribution. Sodium 3-allyloxy-2-hydroxy-1-propanesulfonate (HAPS) caused no fetal malformations and did not influence adversely the occurrence of variations.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL developmental toxicity: 1000 mg/kg bw/day

Executive summary:

Groups of 24 sperm-positive female Hsd. Brl. Han: WIST Rats were treated with Sodium 3-allyloxy-2-hydroxy-1-propanesulfonate (HAPS) by oral administration at three dose levels of 100, 300 and 1000 mg/kg bw/day and one control group of 22 sperm positive females from day 6 up to and including day 19 post coitum daily. The control animals were given the vehicle (aqua purificata) alone. The treatment volume was 5 mL/kg/bw.

Formulation analytics (checking of homogeneity and achieved concentrations of the test item in the dosage forms) was performed two times during the treatment period using a validated HPLC/UV method. The measured concentrations of the Sodium 3-allyloxy-2-hydroxy-1-propanesulfonate (HAPS) formulations varied between 96 and 101 percent of the nominal concentration. During the study animals were checked for mortality and clinical signs. Body weight and food consumption of the dams were also recorded. The day of detection of sperm in the vaginal smear of femaleswas regarded as day 0 of gestation.

A Caesarean section and gross pathology were performed on gestational day 20. Organs of the dams were examined macroscopically.

The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The placentas were weighed and examined externally.The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method.After double staining, the skeletons were examined by means of a dissecting microscope.All abnormalities found during the fetal examinations were recorded.

Results

 In total,the number of evaluated litters was 84 (18 in the control and 22 each in the 100, 300 and 1000 mg/kg bw/day dose groups).

 Clinical Symptoms, Mortality, Necropsy

There was no mortality and were no treatment related clinical signs and necropsy findings observed. 

 Body weight, food consumption

There were no differences indicated attributed to the test item.

Intrauterineparameters

No dose related increase in pre- and post-implantation loss was indicated. The mean number of viable fetuses and their sex distribution was similar in all groups.

Fetal- and placental weight

There were no treatment related differences observed in the fetal- , placental- as well as relative placental weight.

Fetal examination

The number of litters with malformed fetuses was two in the 100 and three in the 1000 mg/kg bw/day groups. There were no malformations found in the control and 300 mg/kg bw/day group.

Malformations:

At external examination two fetuses were found with short tail and one of them with hypoplastic pollex (not proved at skeletal examination) in the 100 mg/kg bw/day dose group. Both of these fetuses had multiple malformed vertebrae and in addition one of them had fused ribs. In this group a third fetus had also fused ribs and multiple malformations of the thoracic vertebrae.In the high dose group two fetuses were found with bent scapula or/and ulna or/and slightly shorter femur. A third fetus in the high dose group had a bipartite thoracic vertebra with dumb-bell shaped cartilage. All of the malformations found in the low and high dose group occur sporadically also in control rat fetuses according to the Background pregnancy and fetal data of Toxi-Coop Zrt. (Appendix XXIV/A) and occurred with low incidence or without a dose response and were considered to be without a test item relationship in this study.

There were no malformations found at visceral examination.

Variations:

There was no increase indicated in the external and visceral variations.

Statistically significant increase of markedly incomplete ossification of one or more skull bones in the 300 and 1000 mg/kg bw/day dose group (without a statistical significance in the high dose group if the litter incidences evaluated) as well as wavy ribs in the low and high dose (the data were within the historical control level) were considered as non-adverse.