Sodium 3-chloro-2-hydroxypropanesulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

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 species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, the testing laboratory has reproductive historical control data in the Crl:CD(SD) rat.

Sex:

male/female

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

Details on route of administration:

The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
The vehicle and test substance formulations were administered via appropriately sized, flexible, disposable, plastic feeding tubes

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral 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 formulations of all dose groups including control). In addition, samples were analyzed for resuspension homogeneity 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%.

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 post-coitum, 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).

Frequency of treatment:

Once daily, 7 d/w

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

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

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).

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

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

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 male and female was also observed for signs of toxicity approximately 2 hours following dose administration.

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and 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, for males and females. 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:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Blood was collected via the jugular vein using the hand-held restraint method for males and via the retro-orbital sinus following isoflurane anesthesia 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:
- 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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.

- all F0 parental animals found dead, euthanized in extremis, or at the scheduled termination:
According to test guideline

- All remaining females which failed to deliver and the remaining males: According to test guidelines

ORGAN WEIGHTS
- All F0 animals: According to test guideline

HISTOPATHOLOGY: Yes
According to test guideline in 5 animals/sex/group in the control and 1000 mg/kg bw/day groups and in all animals dying spontaneously

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.

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.

Ambulatory and total counts measured during motor activity assessments were analyzed by sex and session, with a RMANOVA (SAS version 9.2). Factors in the model included treatment group (TRT), time interval (TIME), and the interaction of time interval and treatment group (TRT*TIME). The SAS® procedure PROC MIXED was used for analysis with the random effect of animal included as the repeated measurement. The covariance structure across time was selected by comparing Akaike’s Information Criterion (AIC).
The monotonic dose-response relationship was evaluated using sequential linear trend tests based on ordinal spacing of dosage levels. The linear dose by time interaction (LinTrt*Time) was evaluatedusing trend tests on treatment means were performed at the 0.05 level for each time interval or across the pooled time intervals by session only.
Nonmonotonic dose responses were evaluated whenever no significant linear trends were detected but TRT and/or TRT*TIME interaction was significant at the 0.01 level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All F0 males and females in the test substance-treated groups survived to the scheduled necropsies. 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:

effects observed, non-treatment-related

Description (incidence and severity):

A significantly (p < 0.01) higher mean body weight gain was noted in F0 males in the 500 mg/kg/day group during Study Days 7–13 compared to the control group. However, this difference was transient and not of sufficient magnitude to affect the mean body weight gains during the pre-mating (Study Days 0–13) and entire treatment (Study Days 0–27) periods, or mean body weights at this dosage level, and did not occur in a dose-related manner. Therefore, the higher mean body weight gain noted in the 500 mg/kg/day group was not considered test substance-related.

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:

effects observed, treatment-related

Description (incidence and severity):

A significantly (p < 0.05) lower (15.5%) mean platelet count was noted for F0 males in the 1000 mg/kg/day group compared to the control group. In the absence of other noteworthy changes in hematology and coagulation parameters at this dosage level, the difference was considered to be the result of normal biological variation and of no toxicological significance.

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:

effects observed, non-treatment-related

Description (incidence and severity):

A significantly (p < 0.05) higher mean relative (to brain weight) left testis weight was noted in the 1000 mg/kg/day group F0 males compared to the control. No histologic correlates were detected to account for the higher mean testis weight. In addition, the mean absolute left testis and brain weights, and left testis to brain weight ratio were generally within the testing laboratory historical control data ranges. The higher mean left testis weight in the 1000 mg/kg/day group F0 males can be partially explained by the data showing that 1 control group male had a left testis weight approximately 26% lower than the mean for that group, and was lower (outside of mean ± 2 S.D.) compared to the testing laboratory historical control data. Therefore, the higher mean left testis weight relative to brain weight in the 1000 mg/kg/day group F0 males was not considered to be test substance-related.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males at any dosage level. The mean lengths of estrous cycles in F0 females at any dose level were comparable to the control.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

Parental generation

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 including the highest dose level tested.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Analytical verification of the test item concentrations

No test substance was detected in the analyzed vehicle administered to the control group.

The concentrations analysed in the first, third and last formulations of 250, 500 and 1000 mg/kg bw/day groups were in agreement with the target concentrations. Mean accuracies were 86.8%, 96.9% and 109% for the 250 mg/kg bw/day group (n = 6, 2 and 2, respectively), 88.3%, 95.4% and 109% for the 500 mg/kg bw/day group (n=2) and 88.4%, 99.7% and 108% for the 1000 m/kg bw/day group (n = 6, 2 and 2, respectively). The formulations of 250 and 1000 mg/kg bw/day groups were homogeneous (i.e. relative standard deviation 6.8% and 6.6%, respectively).

Formulations at the entire range were stable following 10 days of refrigerated storage (i.e. relative difference ≤ 10%).

Applicant's summary and conclusion

Conclusions:

In an oral OECD422 study with rats conducted in accordance with GLP principles, the NOAEL was determined to be 1000 mg/kg bw/day, based on the absence of adverse effects up to and including the highest dose level.