Sodium 3-(allyloxy)-2-hydroxypropanesulphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.11.2012 - 03.05.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: charls River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 274 - 309 g (males); 174 - 199 g (females)
- Fasting period before study: no
- Housing: males in groups of three, females individual
- Diet : Mainenance diet for rats and mice, No. 1324 TPF, ad libitum
- Water : sterilised community tap water, ad libitum
- Acclimation period: 9-10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item has been delivered as a 35,2% aqueous solution and a relative density of 1,17 g/cm3. Dosing referred to the effective content of the active ingredient (HAPS). The raw solution also contained 3,7% NaOH increasing the pH up to 13,2, causing severe corrosive effects during application. Thus, the composition was neutralised to a pH of 7 with concentrated HCl. For preparation of the application solutions the neutralised test solution was diluted with autoclaved community tap water. The test item preparation was intended for an application volume of 4 ml per kg body weight.
All material data sheets were stored with the raw data.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 deays
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Males were dosed daily for 42 to 57 days, including the day before the scheduled termination of the in-life phase. This included two weeks of dosing prior to mating and continued throughout the mating period until approximately four weeks post-mating. Females were dosed two weeks prior to mating, covering at least twocomplete oestrous cycles, the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled termination of the in-life phase. Therefore the duration of the study following acclimatisation depended on the female performance and was at least 47 days: 14 days pre-mating, up to 14 days until mating, an average of 21 days of gestation, and between 8 and 14 days of lactation.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

62.5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes

Positive control:

no

Examinations

Parental animals: Observations and examinations:

1. Viability / fatalities Daily
2. General clinical signs / behaviour Daily
3. Body weight Once weekly (including once before beginning
of application)
4. Group/Individual food consumption Once weekly (including once before beginning
of application)
5. Group/Individual water consumption Once weekly (including once before beginning
of application)

Oestrous cyclicity (parental animals):

yes

Sperm parameters (parental animals):

yes

Litter observations:

yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues [ovaries, testes, epididymis of the highest dose group and the control group] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

- External examinations: Yes: all per litter
- Skeletal examinations: Yes

Statistics:

Statistical Analysis
Spread sheet calculations were performed using Microsoft® Excel® 2011 for Mac.
Food- and water consumption of male animals were documented sorted by experimental groups, whereas the body weight was documented for each animal individually. Body weight, food- and water consumption, litter size and litter weight were documented for each female animal individually.
Descriptive statistics
The arithmetic mean, standard deviation and median were calculated for all grouped numerical data originating from monitoring the body weight, food- and water consumption, organ weights (gross pathology) and litter size and weight (for details see appendix). Where appropriate, detailed column statistics were applied (minimum / maximum data, 25% quantiles, standard error, upper and lower confidence interval 95%).
Inductive statistics
If appropriate, the respective test item groups were compared to the vehicle group by assessing statistical significance using a two-tailed unpaired Student´s t-test. For allcalculations, the significance level was set to 0,05.

Reproductive indices:

examination of reproductive organs

Offspring viability indices:

bodyweight
sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mild discomfort throughout the whole application period was observed for the male animals treated with the high dose of the test item (wiping of nose and mouth through the cage bedding, salivation after application, bleeding of mucous membranes at nose and mouth, respiratory sounds). Due to the solely appearance of the symptoms in the animals treated with the high dose of the test item, it could be estimated with high probability that the test item induced slight irritating effects after application of the highest concentration. Hence, a test item related effect could not be excluded.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Due to the high incidence and the exclusive occurrence within the animals treated with the high dose, a test item related effect could not be excluded. Death could be a result of reflux after gavage dosing leading to an accidental aspiration of dose formulation. Even very small
amounts of irritant test formulations can result in life-threatening respiratory symptoms and death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences were observed between all test item treated male animals and the vehicle control animals. Generally, no signs for a direct test item related effect on the body weight of the female animals could be observed during the first two exposure weeks. Nonetheless, a test item related effect at a later time point could be masked by the variances in body weight resulting from the different number of females achieving pregnancy per dose group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences in food consumption could be observed for all test item treated animals throughout the whole in-life phase. Individual fluctuations were most likely effects of natural origin. On the contrary, an increased water intake of all animals (male and female) treated with the highest dose of the test item could be observed throughout the whole in-life phase when compared to their respective vehicle control animals. Additionally, single fluctuations were observed for the male animals of the medium dose group.
In summary, no significant differences in food consumption could be observed for the test item treated animals throughout the whole in-life phase. Individual fluctuations were most likely effects of natural origin.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Summarised, an increased water intake of all animals (male and female) treated with the highest dose of the test item could be observed throughout the whole in-life phase when compared to their respective vehicle control animals. Additionally, single fluctuations were observed for the male animals of the medium dose group.

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A minimal to slight ovarian hypertrophy/hyperplasia characterised by the presence of many, partly cystic corpora lutea, several tertiary follicles and an increase in the number of interstitial cells was noted in the female animals treated with the high dose of the test item. Although these findings could be due to the infertile state and estrus cycle of the ovaries, a relationship to the treatment with the test item could not excluded.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Effects of the spermatogenesis may not have had an adequate time to become evident (such as reduced sperm counts affecting the fertility), as chemical exposure did not cover a complete cycle of spermatogenesis in male test animals.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Regarding the reproduction and developmental parameters gathered in this study, the test item prevented or significantly reduced the achievement of pregnancy in all tested dose levels. The presence of corpora lutea after first pairing indicated that an implantation of the zygote took place but embryonic development did not occur or was aborted during the first days of gestation. The absence of corpora lutea after second pairing then indicated that, with prolonged dosing the implantation of the zygote or the ovarian maturation were impaired by the test item. Nonetheless, based on the results of the study a specific physiological cause of the toxic effect could not be identified.

Effect levels (P0)

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: lack of achieving pregnancy

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

62.5 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

uterus

Treatment related:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Offspring was present only in the low dose group and in the control group.

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

65.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

62.5 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Applicant's summary and conclusion

Conclusions:

A daily oral administration of the test item Sodium 3-(allyloxy)-2- hydroxypropanesulphonate (HAPS) to male Wistar rats at dose levels of 62,5 mg, 250 mg and 1000 mg/kg body weight over a time period of 42 to 57 days did not produce any pathological evidence for toxic effects on the reproduction performance of male rats.
However, effects of the spermatogenesis may not have had an adequate time to become evident (such as reduced sperm counts affecting the fertility), as chemical exposure did not cover a complete cycle of spermatogenesis in male test animals. Therefore, and due to the lack of pregnancies in the
test item treated female animals, an effect of the test item on the spermatogenesis could not be excluded.
The study has shown that a daily oral administration of the test item at a dosage of 250 mg/kg body weight results in a prevention of pregnancies in female Wistar rats. At a dosage of 62,5 mg/kg, this effect was still apparent in the majority of the animals of this group. Five of 12 animals were able to achieve pregnancy but only 2 of these animals had a normal litter size and development. Hence, a daily oral administration of the test item to female Wistar rats at dose levels above 62,5 mg/kg body weight over a time period of 47 to 76 days produced severe pathological evidence for toxic effects on the reproductionv performanceof female rats regarding the achievement of pregnancy, litter size and survival rate of pups. It must therefore be assumed that the NOAEL regarding reproduction and development of the test item is significantly below the herein used lowest dose of 62,5 mg/kg.

Executive summary:

In the present study toxic effects of the test item Sodium 3-(allyloxy)-2- hydroxypropanesulphonate (HAPS) at a maximum dose of 1000 mg/kg body weight on the development and reproduction of Wistar rats after oral administration were under examination.

General clinical signs

Mild discomfort throughout the whole application period was observed for the male animals treated with the high dose of the test item (wiping of nose and mouth through the cage bedding, salivation after application, bleeding of mucous membranes at nose and mouth, respiratory sounds). Due to the solely appearance of the symptoms in the animals treated with the high dose of the test item, it could be estimated with high probability that the test item induced slight irritating effects after application of the highest concentration. Hence, a test item related effect could not be excluded.

Body weight, food and water consumption

No significant differences were observed between all test item treated male animals and the vehicle control animals. Generally, no signs for a direct test item related effect on the body weight of the female animals could be observed during the first two exposure weeks. Nonetheless, a test item related effect at a later time point could be masked by the variances in body weight resulting from the different number of females achieving pregnancy per dose group.

No significant differences in food consumption could be observed for all test item treated animals throughout the whole in-life phase. Individual fluctuations were most likely effects of natural origin. On the contrary, an increased water intake of all animals (male and female) phase when compared to their respective vehicle control animals. Additionally, single fluctuations were observed for the male animals of the medium dose group.

Necropsy

No test item related prevalent findings were observed during necropsy, neither for male nor for female animals. None of the irregular findings detected at the necropsy could be related to the administration of the test item. They were regarded to be spontaneous in nature.

Histology

The ovaries, testes, and epididymis from a total of 51 adult rats (high dose: 10 male/ 6 female; satellite group: 11 female; vehicle control: 12 male/ 12 female) and all other organs showing macroscopic lesions were examined histopathologically. The morphology of the ovaries of the infertile treated females of the high dose group (including animals of the satellite group) was slightly different from those examined in the dams of the vehicle control group. In the main, a minimal to slight ovarian hypertrophy/hyperplasia characterised by the presence of many, partly cystic corpora lutea, several tertiary follicles and an increase in the number of interstitial cells was noted in the treated females. These findings may be due to the infertile state and estrus cycle of the ovaries. However, a relationship to the treatment with the test item could not excluded. All other microscopic findings recorded in the reproductive organs (epididymides, testes, ovaries, uterus horn and some other macroscopic alterations) of the examined animals were considered to be due to the actual estrus cycle or were spontaneous in nature. These findings were within the normal background pathology commonly seen in rats of this age. The differences noted were regarded as random events. After consultation with the Sponsor, an examination of the medium and low dose animals was not performed.

Reproduction and Development

Regarding the reproduction and developmental parameters gathered in this study, the test item prevented or significantly reduced the achievement of pregnancy in all tested dose levels. The presence of corpora lutea after first pairing indicated that an implantation of the zygote took place but embryonic development did not occur or was aborted during the first days of gestation. The absence of corpora lutea after second pairing then indicated that, with prolonged dosing the implantation of the zygote or the ovarian maturation were impaired by the test item. Nonetheless, based on the results of the study a specific physiological cause of the toxic effect could not be identified.