Sodium hydrogensulphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from 2010-04-22 to 2010-08-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study has been performed in compliance with the: Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted on November 26th, 1997 by decision of the OECD Council [C (97)186/Final]. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). There were no circumstances that may have affected the quality or integrity of the data.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TYPES OF QA INSPECTIONS: study plan; study based: test system, test item, dose preparation, raw data, body weight, treatment; process based: work up formulations analysis, histotechnique, appendix formulations analysis, appendix histopathology, report

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium sulphate

EC Number:

231-820-9

EC Name:

Sodium sulphate

Cas Number:

7757-82-6

Molecular formula:

H2O4S.2Na

IUPAC Name:

disodium sulfate

Details on test material:

Description: White Powder
Batch Number: 20091001
Molecular Weight: 142.04 g/mol
Purity: 99.5%
Expiry Date (Retest Date): 30-Sep-2010
Storage Conditions: Room Temperature (20 ± 5 °C)
Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask).

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Animals: Rat, RccHan: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V.
Kreuzelweg 53
5961 NM Horst / Netherlands
Number of Animals: 40 males: 10 per group
40 females: 10 per group
Age (at Start of Treatment): 11 weeks
Body Weight Range
(at Start of Treatment): Males: 292 to 329 g
Females: 182 to 223 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 – 73.5%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch nos. 83/09 and 22/10).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Sodium Sulphate was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.


STORAGE OF DOSE FORMULATIONS

Dose formulations were stored at room temperature (20 ± 5 °C) in brown glass beakers.


TREATMENT

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group); Group 2: 100 mg/kg/day; Group 3: 300 mg/kg/day; Group 4: 1000 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study C79092 (non-GLP), using dose levels of 100, 300 and 1000 mg/kg/day, resulting in a NOEL of 300 mg/kg/day.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males (4 weeks); females (approximately 7 weeks)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs and 7 days). During the second last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to a conductivity detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.

The test item recoveries were found to be in the range of 86.7% to 97.4% with reference to the nominal concentration. The maximum variation from the mean of homogeneity samples which were taken from the top, middle and bottom of the samples, ranged from 0.5% to 4.7%. The stability of the application formulations was tested in samples taken four hours and seven days after preparation and stored at room temperature. The variation values ranged from 0.2% to 3.2% from the time-zero value.

Duration of treatment / exposure:

Males: 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Acclimatization: 7 days (males and females)
First Test Item Administration: Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 11 days (males); 11 days maximum (females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day before sacrifice (males); on day 3 post partum (females)
Necropsy: After a minimum of 28 days treatment (males); on day 4 post partum (females)

Examinations

Observations and examinations performed and frequency:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males (weekly during pre-pairing and after pairing periods.); females (pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum). No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.

Sacrifice and pathology:

TERMINATION OF THE STUDY

Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum.

When birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.


NECROPSY

At the scheduled sacrifice, all parent animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes at the scheduled necropsy.

For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.


ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.


TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.



HISTOTECHNIQUE

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testes were stained by PAS-hematoxylin.


HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females (nos. 48, 49, 56 and 59) that did not give birth. Microscopic examination of the reproductive organs of all infertile males (nos. 8, 9, 16 and 19) was made.

A histopathology peer review was performed by the Pathology Department (Harlan Laboratories Ltd., Itingen / Switzerland)

Other examinations:

LITTER OBSERVATIONS

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 1 and 4 post partum.


TERMINATION AND NECROPSY OF OFFSPRING

Pups were sacrificed on day 4 post partum.

At the scheduled sacrifice, all pups were killed by an injection of sodium pentobarbital.

All pups were examined macroscopically for any structural changes at the scheduled necropsy.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction data, macroscopical findings and organ weight:
- 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 were applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

1 IN-LIVE DATA - PARENTAL ANIMALS

1.1 CLINICAL SIGNS OR OBSERVATIONS

All animals survived until the scheduled necropsy and no clinical signs were noted during the entire duration of the study.

1.2 FOOD CONSUMPTION OF MALES

Pre-pairing and After Pairing Periods

Mean food consumption was not affected by the treatment with the test item at any dose level.

In order of ascending dose levels, the overall differences in food consumption were: ±0.0%, +2.4% and -0.8% during pre-pairing period and -1.7%, -7.4% and -2.9% during the after pairing period (percentages refer to the respective values of the control group).

1.3 FOOD CONSUMPTION OF FEMALES

Pre-pairing, Gestation and Lactation Periods

No statistically significant alterations of mean food consumption were observed at any dose level when compared to the respective values in the control group.

In order of ascending dose levels, the overall differences in food consumption were: +6.0%, ±0.0% and -3.3% during the pre-pairing period, +1.6%, +2.0% and -3.6% during the gestation period and -3.7%, +4.0% and -5.6% during the lactation period (percentages refer to the respective values of the control group).

1.4 BODY WEIGHTS OF MALES

Pre-pairing, Pairing and After Pairing Periods

No significant changes were observed in mean body weight and mean body weight gain.

In the order of ascending dose levels, the overall mean body weight gains were: +11%, +9%, +13% and +11% during the pre-pairing period, +6%, +5%, +5% and +4% during the pairing period and +4%, +4%, +4% and +3% during the after pairing period (percentages refer to the respective time intervals).

1.5 BODY WEIGHTS OF FEMALES

Pre-pairing, Gestation and Lactation Periods

No significant alterations of mean body weights and mean body weight gain were observed at any dose level when compared to the respective values in the control group.

In the order of ascending dose levels, the overall mean body weight gain was +10%, +11%, +12% and +9% during the pre-pairing period and +57%, +59%, +57% and +57% during the gestation period and +5%, +4%, +6% and +6% during the lactation (percentages refer to the respective time intervals).


2 REPRODUCTION AND BREEDING DATA

2.1 MATING PERFORMANCE AND FERTILITY

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.3, 4.3, 3.3 and 3.1 days in order of ascending dose level. The median precoital time was 3, 4, 3 and 3 days in order of ascending dose level.

Two females each in groups 1 and 2 were not pregnant. Thus the fertility indices were 80.0%, 80.0%, 100.0% and 100.0% in groups 1, 2, 3 and 4.

2.2 DURATION OF GESTATION

The mean duration of gestation was unaffected by treatment with the test item. Mean duration of gestation was 21.4, 21.5, 21.7 and 21.5 days, in order of ascending dose level.

2.3 CORPORA LUTEA COUNT

The mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.6, 14.1, 14.2 and 14.1 in order of ascending dose level) and gave no indication of a test item-related effect.

2.4 IMPLANTATION RATE AND POST-IMPLANTATION LOSS

The mean number of implantations per dam and the post-implantation losses were unaffected by treatment with the test item.

The mean numbers of implantations per litter were 13.3, 13.5, 14.0 and 13.3 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was 10.4, 5.6, 10.0 and 3.8% in order of ascending dose level.

2.5 LITTER SIZE AT FIRST LITTER CHECK

The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was 11.9, 12.8, 12.6 and 12.8 in order of ascending dose level. The number of pups found dead at first litter check was two pups each in the control group and in group 2.

2.6 POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

The total number of pup loss during the first four days was only one pup in the control group on day 3. Therefore the viability indices were 98.9% in the control group and 100.0%, in groups 2, 3 and 4.


3 TERMINAL FINDINGS - PARENTAL ANIMALS

3.1 ORGAN WEIGHTS

In males, weights (absolute and relative to body weight) of testes and epididymides were not affected by the treatment with the test item in any groups.

3.2 MACROSCOPICAL FINDINGS

Males

In group 4, three males were noted to have the pelvis of right kidney dilated.

In group 3, no abnormal findings were noted.

In group 2, one male was noted to have both testes and epididymides reduced in size.

In control group, one male was noted to have a pelvic dilation and enlargement of left kidney and another male to have both testes and epididymides reduced in size.

Type and frequencies of these findings did not give an indication of a test item-related effect.

Females

No abnormal findings were noted in females.

3.3 HISTOPATHOLOGY FINDINGS

All findings recorded were within the range of normal background lesions which may be recorded in rats of this strain and age.

In testes of animal no. 9 in group 1 and no. 19 in group 2, marked to severe tubular atrophy was found bilaterally and this finding was considered to be associated with their infertility. In the above 2 animals, aspermia and cellular debris in epididymides which were considered to be associated with the tubular atrophy of testes were also observed. No microscopic findings were found in other reproductive organs of these animals, reproductive organs of other infertile males (nos. 8 and 16) and ovaries of females (nos. 48, 49, 56 and 59) that did not give birth.

Treatment with the test item did not reaveal effects on the completeness of stages or cell populations. There was no indication for maturation arrest or any other degenerative type.

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: NOEL = highest dose tested

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated	10	10	10	10
Number of pregnant females (A)	8	8	10	10
Number of females which reared their pups until day 4 post partum	8	8	10	10

(A)  Female Nos. 48 and 49 in group 1 and nos. 56 and 59 in group 2 were not pregnant.

Applicant's summary and conclusion

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Sodium Sulphate to rats. Sodium Sulphate was administered in highly purified water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. Sodium Sulphate was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

In absence of any effect the general NOEL (No Observed Effect Level) was established at 1000 mg/kg/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Sodium Sulphate on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

Four groups of 10 males and 10 females were treated by gavage with Sodium Sulphate once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

 

The following dose levels were used:

                       Group 1:                0 mg/kg body weight/day (control group)

                       Group 2             100 mg/kg body weight/day

                       Group 3:            300 mg/kg body weight/day

                       Group 4           1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 

PARENTAL ANIMAL

 

General Tolerability

 

All animals survived until the scheduled necropsy and no clinical sign was observed during the study.

 

Food Consumption

 

Mean food consumption was not affected by treatment with the test item in males and females. 

 

Body Weights

 

No test item-related effects were observed in mean body weight and mean body weight gain of males and females.

 

Reproductive Data

 

Mating performance, fertility index and conception rate were not affected by treatment with the test item. 

 

The mean number of corpora lutea, the mean number of implantations per dam, and the post-implantation losses were unaffected by treatment with the test item. The mean duration of gestation was also unaffected by treatment with the test item.

 

Organ Weights

 

Mean weight of testes and epididymides were not affected by the treatment with the test item.

 

Macroscopical Findings and Histopathological Examinations

 

All organs and tissues examined did not reveal any macroscopic nor microscopic changes related to the treatment with the test item. 

 

LITTER DATA - F1 PUPS

 

The number of live pups at first litter check and the mean litter size was unaffected by treatment with the test item. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item. Mean pup weights and weight gains were also not considered to be affected.