Sodium 3-chloro-2-hydroxypropanesulphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

(combined repeated dose and reproduction / developmental screening)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 2016 - 11 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (March 1996)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-chloro-2-hydroxypropanesulphonate

EC Number:

204-807-0

EC Name:

Sodium 3-chloro-2-hydroxypropanesulphonate

Cas Number:

126-83-0

Molecular formula:

C3H7ClO4S.Na

IUPAC Name:

sodium 3-chloro-2-hydroxypropanesulphonate

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Age at study initiation: Approximately 10 weeks.
- Weight at study initiation: 307-394 gr (males) or 201-291 gr (females)
- Fasting period before study: no. All males and females (including those not selected for clinical pathology evaluation) were fasted prior to clinical pathology blood collection when food, but not water, was withheld.
- Housing:
Pre-mating: Animals were housed in groups of 2-3 animals/sex/cage in solid-bottom cages.
Mating: Females were caged together with males on a one-to-one-basis.
Post-mating: Males and females were individually housed until the schedule necropsy.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (PMI Nutrition International, LLC Certified Rodent LabDiet® 5002), except o/n before sacrifice.
- Water: Free access to tap water.
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26 (actual range during study 21.8 - 23.1)
- Humidity (%): 30 - 70 (actual range during study 33.0 - 44.2)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 November 2016 to 20 February 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle suspension was prepared approximately weekly for administration to the control group and for preparation of the test substance formulations. Following formulation, the pH of the vehicle was adjusted to a pH of 3.5 ± 0.4 using 2 N or 5 N HCl. Aliquots were prepared for daily dispensation to the control group and stored refrigerated (2°C to 8°C). The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (2°C to 8°C). The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE: 0.5% carboxymethylcellulose (CMC) and 0.1% Tween® 80 in deionized water, pH 3.5
- Justification for use and choice of vehicle (if other than water): Vehicle is accepted in international guidelines and use of vehicle resulted in formulations acceptable for administration.
- Concentration in vehicle: 25, 50 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were collected at the test facility and analyzed for homogeneity (first 250 and 1000 mg/kg bw/day dosing formulations) and concentration (first, third and last dosing for mulations of all dose groups including control). In addition, samples were analyzed for resuspension homogen eity and stability (the first 250 and 1000 mg/kg bw/day dosing suspensions following refrigerated (2°C to 8°C) storage for 10 days).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the relative standard deviation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After 14 days of unsuccessful pairing two females (250 mg/kg bw/day) who had not shown evidence of mating was separated from their males.
- After successful mating each pregnant female was caged individually.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 50-63 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 4 days of lactation. Females with no evidence of mating were dosed for 52 days through the day prior to euthanasia (Postcohabitation Day 25).
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 28 days
Females: 50-63 days
Pups: 13 days

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on results of a 10-Day dose range finding study. In this study, the test substance was administered to males and females at dosage levels of 100, 250 and 500 mg/kg/day. The test substance was well tolerated in both males and females, with no significant adverse effects at any dosage level. Therefore, a high-dosage level of 1000 mg/kg/day was used in the current study as the limit dose, which was expected to produce signs of toxicity, without causing mortality. Dosage levels of 250 and 500 mg/kg/day were selected as the low- and mid-dosage levels to establish a dose response and a no-observed-adverse-effect level (NOAEL).

Parturition:
During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Culling:
To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily and detailed physical examinations were conducted weekly (prior to dose administration during the treatment period). Each female was also observed for signs of toxicity approximately 2 hours following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on days 0 (when possible), 1, 7, 10, 13 and 14.

FOOD CONSUMPTION: Yes.
- Weekly. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on days 1, 4, 7, 10 and 13 of lactation.

FOOD EFFICIENCY: No.
WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane) for females
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (overnight). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guideline

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and
the selected females were tested during lactation (LD13) (all before blood sampling).
- Dose groups that were examined: All 5 animals/sex/group
- Battery of functions tested: According to test guideline. Home cage observations, open field observations, sensory observations and neuromuscular observations were performed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13.
- Body weights: Live pups were weighed on days 1, 4, 7, 10 and 13 of lactation.
- Sex: Sex was determined for all pups on days 0, 4 and 13 of lactation (by assessment of the anogenital distance).
- Anogenital distance: PND 1, the absolute distance and the absolute distance relative to the cube root of body weight were reported for each pup.
- Assessment of Areolas/Nipple anlagen: On PND13 in all F1 male pups.
- Thyroid hormone analysis (Thyroxine (T4)): PND13 males and females (1 pup/sex/litter)

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.
- Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor.

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Indices:

Reproductive indices; For each group, the following calculations were performed:
For each group, the following calculations were performed:
- Male (Female) Mating Index (%): No. of males (Females) with evidence of mating (or confirmed pregnant)/Total No. of males (females) used for mating x 100
- Male Fertility Index (%): No. of males siring a litter/Total No. of males used for mating x 100
- Male Copulation Index (%): No. of males siring a litter/ No. of males with evidence of mating (or females with confirmed pregnancy) x 100
- Female Fertility index: No. of females with confirmed pregnancy/No. of females used for mating x 100
- Female Conception Index (%): No. of females with confirmed pregnancy /No. of females with evidence of mating x 100

Offspring indices:
Mean Live Litter Size: Total No. of viable pups on PND 0/No. of litters with viable pups PND 0
Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter): Sum of (viable pups per litter on PND 0 or PND 4/No. of Pups Born Per Litter)/No. of litters per group x 100
Postnatal Survival for All Other Intervals (% Per Litter): Sum of (viable pups per litter at end of interval N/viable pup per litter at start of interval N)/No. of litters per group x 100
where N = PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–10, 10–13, birth to PND 4, and 4 (post-selection)–13

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the control group, one F0 female was found dead on Gestation Day 12; no clinical observations or noteworthy changes in body weight were noted for this female prior to death. Following macroscopic and microscopic examinations, the cause of death was determined to be an acute pericardial hemorrhage. An additional F0 Female in the control group was euthanized in extremis on Lactation Day 0 due to poor clinical condition, including observations of a cool and pale body and extremities at the daily examinations on the day prior to and/or the day of euthanasia. Following macroscopic and microscopic examinations, the cause of the moribund condition of this female was uterine hemorrhage, which was consistent with uterine torsion noted at the gross examination. All other control group animals survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

During open field oberservations significantly (p < 0.05 or p < 0.01) longer mean times to first step (0.6 and 0.7 seconds) were noted for F0 females in the 250 and 500 mg/kg/day groups, respectively, compared to the control group (0.3 seconds). In the absence of a dose-related response, the differences were not considered test substance-related. In addition, the mean time to first step values in the 250 and 500 mg/kg/day groups were within the range of values (0.22 to 1.15 seconds) in the testing laboratory historical control data (version 2016.02).

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

pre-implantation loss was not examined.

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

Description (incidence and severity):

No statistically significant differences were noted between the control and test substance-treated groups. One male and female mating pair in the 250 mg/kg/day group did not produce a litter.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No toxicity was observed up to and inculding the highest dose level tested.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

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

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Anogenital distance and thyroid hormones values in F1 pups and areolae/nipple anlagen in the F1 males were unaffected by parental administration of the test substance. There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dosage level on PND 13.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to and inculding the highest dose level tested.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In an oral OECD 422 screening study with rats performed in accordance with GLP principles, the parental NOAEL and the developmental NOAEL were derived to be 1000 mg/kg bw/day, based on the absence of adverse effects up to and including the highest dose level tested.