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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start and completion dates: 22 April 2010 to 30 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD guideline 421 (OECD guideline for testing of chemicals 421, “Reproduction/Developmental Toxicity Screening Test”, adapted by the Council on 27th July 1995.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
To generate preliminary information using the OECD 421 "Reproduction/Developmental Toxicity Screening Test" concerning the effects of Sodium Sulphate on male and female reproductive performance such as gonadal function, mating behaviour, conception and parturition. Recognised by international guidelines as a recommended test system.

Test material

Constituent 1
Chemical structure
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
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Supplied by the Sponsor. Treuhandgemeinschaft Deutscher Chemiefasererzeuger GmbH (TDC)
- Purity: 99.5%
- Expiry Date (Retest Date): 30 Sep 2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Application formulations were found to be homogeneously prepared with sufficient formulation stability under study storage conditions.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Soluble in highly purified water at formulated concentrations of 100, 300 & 1000 mg/kg bw/day. Stability of test formulations confirmed analytically.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Formulated to concentration in purified water

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: None

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Rat, RccHan: WIST(SPF)
Details on species / strain selection:
Rat, RccHan: WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst, Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 292 to 329 g, Females: 182 to 223 g
- Fasting period before study: No
- Housing: 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.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (ad libitum): Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst, Switzerland (Batch No's. 83/09 and 22/10.
- Water (ad libitum): Community tap-water from Füllinsdorf was available in water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 73.5%
- Air changes (per hr): Air-conditioned with 10 - 15 air changes/hr
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle

IN-LIFE DATES: 29-Apr-2010 (test item administration) to 30-Aug-2010 (experimental completion)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared weekly. 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. On obtaining an homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Dose formulations were stored at room temperature in brown glass beakers.

VEHICLE
- Purified water
- Concentration in vehicle: 10, 20 and 100 mg/kg dosed at a dose volume of 10 mL/kg bw.
- Amount of vehicle (if gavage): Dose volume of 10 mL/kg with a daily adjustment to actual body weight.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed.
- Proof of pregnancy: If the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dose Formulations:
On treatment day 1, 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 (at 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 Dr. K. Morgenthal (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. Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf, Switzerland.

The linearity of the analytical systems used for sample analyses was demonstrated with a good
relationship between peak areas measured and working standard concentrations. All calibration
points used met the acceptance limit of ±20% variation from the calibration curve derived by
linear regression analysis. The regression coefficients calculated were found to be better than
0.99.

The Sodium Sulphate peak was assigned in sample chromatograms by comparison to that of
working standards. In blank sample chromatograms no peak appeared at the retention time of
Sodium Sulphate and, therefore, the absence of the test item in the control samples was
confirmed.

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.
Results indicate the accurate use of the test item Sodium Sulphate and purified
water as vehicle during this study. Application formulations were found to be homogeneously
prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
Duration of Treatment Period:
Males: 4 weeks
Females: ca. 7 weeks
Frequency of treatment:
Once daily with treatment ending on day 3 post partum for females and on the day before sacrifice for males.
Details on study schedule:
Study Sequence Males Females

Acclimatization 7 days 7 days
First Test Item Administration Day 1 of pre-pairing Day 1 of pre-pairing
Pre-Pairing 14 days 14 days
Pairing 11 days maximum 11 days
Gestation Approximately 21 day -
Treatment Ends On day 3 post partum On day before sacrifice
Necropsy On day 4 post partum After a min. of 28 days treatment


Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Middle dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
40 males: 10 per group
40 females: 10 per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study C79092, using dose levels of 100, 300 and 1000 mg/kg/day, resulting in a NOEL of 300 mg/kg/day.
- Dose selection rationale:
- Rationale for animal assignment: By computer-generated random algorithm.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Viability / Mortality: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs: Once daily, during acclimatization and up to day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION (oral gavage study, not a feeding (diet) study:
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.

Oestrous cyclicity (parental animals):
During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.

During the pairing period, females were housed with sexually mature males (1:1) until evidence
of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter size, live births, sex ratio, still births and any gross anomalies. Pups were weighed individually (without identification) on days 1 and 4 post partum.

GROSS EXAMINATION OF DEAD PUPS: No.
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
SACRIFICE
At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. Parental generation animals were exsanguinated.

GROSS NECROPSY
Pups were sacrificed on day 4 post partum. All pups were examined macroscopically for any structural changes at the scheduled necropsy.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not applicable for the litter (only parental animals).
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 [see References (2)] (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 [see References (3)] (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 [see References (4)] were applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Mating Performance and Fertility:
The median and mean precoital times were unaffected by test item treatment. 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 in groups 1 and two females in group 2 were not pregnant. Thus the fertility indices were 80.0%, (Group 1) 80.0% (Group 2), 100.0% (Group 3) and 100.0% (Group 4).

Fertility index = (Females achieving a pregnancy / Females paired) x 100
Offspring viability indices:
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.

Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) x 100.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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.
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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
In the testes of animal no. 9 (Group 1) and no. 19 (Group 2), marked to severe tubular atrophy was found bilaterally and this finding was considered to be associated with their infertility. In both males, 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).

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.
Reproductive performance:
no effects observed
Description (incidence and severity):
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.

Details on results (P0)

All animals survived until the scheduled necropsy and no clinical signs were noted during the
entire duration of the study.
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).
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).
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).
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).
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.
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.
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.
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.
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.
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.
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.
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 reveal effects on the completeness of stages or cell
populations. There was no indication for maturation arrest or any other degenerative type.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: An absence of any adverse effect, therefore the NOEL was established at 1000 mg/kg/day.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs at first litter check and during lactation were wound on ear or on nose or on mouth in one pup in group 1, in four pups in group 3 and one pup in group 4 and hind foot injured and toes missing in one pup in group 2. None of these findings gave an indication of a test item-related effect.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live pups at first litter check was unaffected by treatment with the test item. The number of pups found dead at first litter check was two pups each in the control group and in group 2.

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.
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
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

At first litter check and during lactation, the clinical signs noted were wound on ear or on nose or
on mouth in one pup in group 1, in four pups in group 3 and one pup in group 4 and hind foot
injured and toes missing in one pup in group 2. None of these findings gave an indication of a
test item-related effect.
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test
item. The proportion of males on day 4 post partum was 48%, 49%, 50% and 45% in order of
ascending dose level.
Mean pup weights were unaffected by treatment with the test item. On day 1 post partum mean
pup weights were 6.1, 6.1, 6.4 and 6.2 g for combined data of male and female pups in order of
ascending dose level. Mean pup weight development during lactation was unaffected by treatment with the test item. Mean pup weights on day 4 post partum were 9.6, 9.4, 9.7 and 9.3 g for combined data of male and female pups in order of ascending dose level.
No abnormal findings were noted at macroscopic examination of the pups.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No findings gave any indication of a test item-related effect.
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Treatment related:
no

Any other information on results incl. tables

Table 1. Summary of food consumption in  P0 male.













PRE-PAIRING PERIOD


Days 1-8    MEAN


ST.DEV. N


Days 8-14   MEAN


ST.DEV.


N


MEAN OF MEANS


Over PRE-PAIRING PERIOD


AFTER PAIRING PERIOD


Days 1-6    MEAN


ST.DEV. N



Group 1


mg/kg


 24.8


 1.0


10


 25.7


 1.6


10


 25.3


 24.2


10



Group 2


mg/kg


 1.7


10


 1.6


10


 25.3


 2.2 10



Group 3


mg/kg


 1.5


10


 2.0


10


 25.9


 2.8 10



Group 4


mg/kg


 2.1


10


 1.7


10


 25.1


 1.4 10



Table 2. Summary of food consuption P0 female
















PRE-PAIRING PERIOD


Days 1-8    MEAN


ST.DEV. N


Days 8-14   MEAN


ST.DEV.


N


MEAN OF MEANS


Over PRE-PAIRING PERIOD


GESTATION PERIOD


Days 0-7    MEAN


ST.DEV. N


Days 7-14   MEAN


ST.DEV. N


Days 14-21 MEAN ST.DEV. N



Group 1


0 mg/kg


 17.2


 1.2


10


 19.3


 1.4


10


 18.2


 22.9


 1.8


8


 24.9


 2.1


8


 26.5


 2.1 8



Group 2


100 mg/kg


 18.2  1.4


10


 20.3  1.2


10


 19.3


 22.6  4.0


8


 25.6  1.4


8


 27.6  1.5


8


 25.2


 31.1  2.7 8



Group 3


300 mg/kg


 17.2  1.6


10


 19.1  1.8


10


 18.2


 22.9  1.5


10


 25.1  1.8


10


 27.7  2.6


10


 25.3


 33.6  4.4 10



Group 4


1000 mg/kg


 16.9  1.0


10


 18.4  1.3


10


 17.6


 21.6  1.4


10


 23.5  1.6


10


 26.6  1.9


10


 23.9


 30.5  4.3 10



MEAN OF MEANS             24.8


Over GESTATION PERIOD (Days 0-21)


LACTATION PERIOD


Days 1-4    MEAN          32.3


                              ST.DEV.        5.5


                              N                 8



*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig. (-)


Table 3. summary of differences in mean food consumption of P0 males













PRE-PAIRING PERIOD


Days 1-8    MEAN


ST.DEV.


N


DIFF (%)*


Days 8-14   MEAN


ST.DEV.


N


DIFF (%)*


MEAN OF MEANS


Over PRE-PAIRING PERIOD


DIFF (%)*


Over PRE-PAIRING PERIOD


AFTER PAIRING PERIOD


Days 1-6    MEAN


ST.DEV.


N


DIFF (%)*



Group 1


mg/kg


 24.8


 1.0


---


 25.7


 1.6


---


---


 24.2


 1.1


10


---



Group 2


mg/kg


 25.1


 1.7


10


+1.2


 25.4


 1.6


10


-1.2


±0.0


 23.8


 2.2


-1.7



Group 3


mg/kg


 25.7


 1.5


10


+3.6


 26.1


 2.0


10


+1.6


+2.4


 22.4


 2.8


-7.4



Group 4


mg/kg


 24.8


 2.1


10


±0.0


 25.4


 1.7


10


-1.2


-0.8


 23.5


 1.4


-2.9



*: Percentage relative to the control group


 


Table 4. Summary of diffrenecs in mean food consumption in P0 Female
















PRE-PAIRING PERIOD


Days 1-8    MEAN


ST.DEV.


N


DIFF (%)*


Days 8-14   MEAN


ST.DEV.


N


DIFF (%)*


MEAN OF MEANS


Over PRE-PAIRING PERIOD


DIFF (%)*


Over PRE-PAIRING PERIOD


GESTATION PERIOD


Days 0-7    MEAN


ST.DEV.


N


DIFF (%)*


Days 7-14   MEAN


ST.DEV.


N


DIFF (%)*


Days 14-21 MEAN ST.DEV.


N


DIFF (%)*



Group 1


mg/kg


 17.2


 1.2


---


 19.3


 1.4


---


---


 22.9


 1.8


---


 24.9


 2.1


---


 26.5


 2.1


8


---



Group 2


mg/kg


 18.2


 1.4


10


+5.8


 20.3


 1.2


10


+5.2


+6.0


 22.6


 4.0


8


-1.3


 25.6


 1.4


8


+2.8


 27.6


 1.5


8


+4.2


 25.2



Group 3


mg/kg


 17.2


 1.6


10


±0.0


 19.1


 1.8


10


-1.0


±0.0


 22.9


 1.5


10


±0.0


 25.1


 1.8


10


+0.8


 27.7


 2.6


10


+4.5


 25.3



Group 4


mg/kg


 16.9


 1.0


10


-1.7


 18.4


 1.3


10


-4.7


-3.3


 21.6


 1.4


10


-5.7


 23.5


 1.6


10


-5.6


 26.6


 1.9


10


+0.4


 23.9



MEAN OF MEANS             24.8



*: Percentage relative to the control group


 


Table 5. Summary of P0 male body weight gain.






































 



Pre-pairing period



Group 1 (0 mg/kg bw/day)



Group 2 (100 mg/kg bw/day)



Group 3 (300 mg/kg bw/day)



Group 4 ( 1000 mg/kg bw/day)



 



Day 1    MEAN ST.DEV.


N


Day 2    MEAN ST.DEV.


N


Day 3    MEAN ST.DEV.


N


Day 4    MEAN ST.DEV.


N


Day 5    MEAN ST.DEV.


N


Day 6    MEAN ST.DEV.


N


Day 7    MEAN ST.DEV.


N


Day 8    MEAN ST.DEV.


N


Day 9    MEAN ST.DEV.


N


Day 10   MEAN ST.DEV.


N


Day 11   MEAN ST.DEV. N



 308  9


10


 314  9


10


 317  9


10


 321


 10


10


 318


 10


10


 326


 11


10


 322


 10


10


 326


 10


10


 330


 10


10


 335


 11


10


 339


 10 10



 307 -


 6


10


 313 -


 6


10


 315 -


 7


10


 319 -


 8


10


 317 -


 7


10


 324 -


 8


10


 320 -


 8


10


 323 -


 9


10


 327 -


 9


10


 329 -


 10


10


 332 -


 10


10



 310 -


 8


10


318 -


 9


10


319 -


 9


10


 325 -


 9


10


 324 -


 9


10


 331 -


 10


10


 329 -


 11


10


 334 -


 11


10


 336 -


 14


10


 340 -


 13


10


 344 -


 12


10



 307 -


 9


10


 317 -


 10


10


 317 -


 9


10


 320 -


 11


10


 319 -


 9


10


 326 -


 10


10


 323 -


 10


10


 328 -


 11


10


 332 -


 12


10


 335 -


 12


10


 338 -


 12


10



 



 


PRE-PAIRING PERIOD


Day 12   MEAN ST.DEV.


N


Day 13   MEAN ST.DEV.


N


Day 14   MEAN ST.DEV. N


PAIRING PERIOD


Day 1    MEAN ST.DEV.


N


Day 2    MEAN ST.DEV.


N


Day 3    MEAN ST.DEV.


N


Day 4    MEAN ST.DEV.


N


Day 5    MEAN ST.DEV.


N


Day 6    MEAN ST.DEV.


N


Day 7    MEAN ST.DEV.


N


Day 8    MEAN ST.DEV. N



 


 


 338


 11


10


 339


 12


10


 344


 11 10


 337


 14


10


 339


 12


10


 342


 12


10


 344


 11


10


 347


 12


10


 351


 11


10


 347


 12


10


 350


 12 10



 


 


332 -


 12


10


 332 -


 12


10


 335 -


 12 10


331 -


 14


10


332 -


 15


10


335 -


 15


10


336 -


 17


10


 339 -


 17


10


342 -


 17


10


 338 -


 18


10


343 -


 17


10



345 -


 13


10


346 -


 14


10


 350 -


 14 10


 345 -


 15


10


 348 -


 14


10


 350 -


 17


10


 352 -


 15


10


355 -


 15


10


 357 -


 15


10


356 -


 16


10


 359 -


 17


10



338  -


 13


10


339  -


 13


10


342  -


 13 10


 338 -


 14


10


340  -


 14


10


341  -


 12


10


343  -


 13


10


 347 -


 12


10


 347 -


 12


10


344  -


 13


10


 347 -


 12


10



Day 9    MEAN ST.DEV.


N


Day 10   MEAN ST.DEV. N


AFTER PAIRING PERIOD


Day 1    MEAN ST.DEV.


N


Day 2    MEAN ST.DEV.


N


Day 3    MEAN ST.DEV.


N


Day 4    MEAN ST.DEV.


N


Day 5    MEAN ST.DEV.


N


Day 6    MEAN ST.DEV. N



 354


 12


10


 356


 12 10


 356


 14


10


 361


 13


10


 362


 13


10


 360


 14


10


 362


 14


10


 371


 14 10



 345 -


 17


10


 348 -


 17 10


 349 -


 19


10


 353 -


 19


10


 355 -


 19


10


 351 -


 19


10


 356 -


 19


10


 362 -


 20


10



 361 -


 18


10


 364 -


 17 10


 366 -


 19


10


370 -


 19


10


371 -


 19


10


 370 -


 19


10


 373 -


 19


10


 380 -


 19


10



 349 -


 12


10


 352 -


 12 10


 351 -


 12


10


355  -


 13


10


356  -


 13


10


 355 -


 13


10


 358 -


 13


10


 363 -


 14


10



 



Table 6. Summary of Female body weight gain 


 















































Periods



Group 1 (0 mg/kg bw/day)



Group 2 (100 mg/kg bw/day)



Group 3 (300 mg/kg bw/day)



Group 4 (1000 mg/kg bw/day)



 



Pre-pairing period


 


 


Day 1    MEAN ST.DEV.


N


Day 2    MEAN ST.DEV.


N


Day 3    MEAN ST.DEV.


N


Day 4    MEAN ST.DEV.


N


Day 5    MEAN ST.DEV.


N


Day 6    MEAN ST.DEV.


N


Day 7    MEAN ST.DEV.


N


Day 8    MEAN ST.DEV.


N


Day 9    MEAN ST.DEV.


N


Day 10   MEAN ST.DEV.


N


Day 11   MEAN ST.DEV. N



 201  8


10


 205  7


10


 203  7


10


 207  8


10


 207  9


10


 209  8


10


 207  8


10


 212


 10


10


 214


 10


10


214      8


10


215      8


10



 198 -


 11


10


 202 -


 9


10


 204 -


 12


10


 207 -


 15


10


 206 -


 12


10


 208 -


 10


10


 208 -


 13


10


 211 -


 13


10


213 -


 13


10


214 -


 11


10


 219 -


 13


10



 198 -


 12


10


203 -


 15


10


204 -


 13


10


 208 -


 14


10


 205 -


 13


10


208 -


 15


10


209 -


 13


10


213 -


 12


10


214 -


 13


10


 214 -


 16


10


 221 -


 14


10



 202 -


 9


10


 206 -


 9


10


 208 -


 9


10


 211 -


 9


10


 208 -


 9


10


 212 -


 10


10


 211 -


 9


10


214  -


 11


10


215  -


 12


10


 215 -


 11


10


 221 -


 11


10



 



 


 


Day 12   MEAN ST.DEV.


N


Day 13   MEAN ST.DEV.


N


Day 14   MEAN ST.DEV. N


PAIRING PERIOD


Day 1    MEAN ST.DEV.


N


Day 2    MEAN ST.DEV.


N


Day 3    MEAN ST.DEV.


N


Day 4    MEAN ST.DEV.


N


Day 5    MEAN ST.DEV.


N


Day 6    MEAN ST.DEV.


N


Day 7    MEAN ST.DEV.


N


Day 8    MEAN ST.DEV. N



 


 


 219 


   9


10


 221


 10


10


 221


   9


10


 227


 3


6


 228


 6


6


 236


 4


3


 238


 8


2


 249


 0


1


 251


 0


1


 253


 0


1


 258


 0 1



 


 


 


 218 -


 15


10


 220 -


 13


10


 219 -


 11 10


 226 -


 13


10


 232 -


 11


7


 235 -


 14


6


 235 -


 1


2


 240 -


 1


2


 239 -


 6


2


 243


 0


1


 245


 0 1



 


 


 221 -


 14


10


220 -


 13


10


221 -


 16 10


 228 -


 13


9


 231 -


 13


8


 230 -


 16


3


 246 -


 0


1


249 -


 0


1


250 -


 0


1


---


---


0


---


---


0



 


 


221 -


 12


10


219  -


 12


10


220  -


 12 10


 228 -


 11


10


 226 -


 12


9


 226 -


 18


3


---


---


0


---


---


0


---


---


0


---


---


0


---


---


0



 



Pairing period


 


Day 9    MEAN ST.DEV.


N


Day 10   MEAN ST.DEV. N


GESTATION PERIOD


Day 0    MEAN ST.DEV.


N


Day 1    MEAN ST.DEV.


N


Day 2    MEAN ST.DEV.


N


Day 3    MEAN ST.DEV.


N


Day 4    MEAN ST.DEV.


N


Day 5    MEAN ST.DEV.


N


Day 6    MEAN ST.DEV.


N


Day 7    MEAN ST.DEV.


N


Day 8    MEAN ST.DEV. N



 263


 0


1


 265


 0 1


 227


 11


8


 231


 12


8


 235


 11


8


 239


 11


8


 244


 11


8


 247


 11


8


 249


 14


8


 253


 13


8


 258


 14 8



 252


 0


1


 251


 0 1


 228 -


 16


8


 234 -


 17


8


 240 -


 17


8


242 -


 16


8


243 -


 17


8


 249 -


 17


8


 254 -


 17


8


 257 -


 15


8


 263 -


 16 8



---


---


0


---


---


0


 228 -


 14


10


 234 -


 13


10


 240 -


 14


10


 244 -


 15


10


 248 -


 14


10


 252 -


 13


10


 256 -


 13


10


 258 -


 13


10


 264 -


 13


10



---


---


0


---


---


0


 227 -


 11


10


 232 -


 13


10


 237 -


 13


10


 239 -


 12


10


 241 -


 11


10


 245 -


 11


10


 249 -


 12


10


 253 -


 12


10


 256 -


 12


10



 



 



Day 20   MEAN ST.DEV.


N


Day 21   MEAN ST.DEV. N


LACTATION PERIOD


Day 1    MEAN ST.DEV.


N


Day 2    MEAN ST.DEV.


N


Day 3    MEAN ST.DEV.


N


Day 4    MEAN ST.DEV. N



 352


 19


8


 362


 23 8


 273


 12


8


 275


 13


8


 280


 14


8


 286


 13 8



 359 -


 22


8


 373 -


 26 8


278 -


 18


8


279 -


 18


8


 281 -


 15


8


 290 -


 19 8



 357 -


 30


10


 369 -


 31 10


 278 -


 15


10


 288 -


 19


10


 285 -


 14


10


 295 -


 18


10



 349 -


 16


10


 364 -


 18 10


 267 -


 11


10


 272 -


 13


10


 277 -


 14


10


 284 -


 11


10



 



*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig. (-)


 


Table 7. Mating performance









































Days of the pairing period.



Group 1


mg/kg


 


 



Group 2


mg/kg


 


 



Group 3


mg/kg


 


 



Group 4


mg/kg


 



1


2


3


4


5


7


11



4


-


 


3


1


1


-


1



-


3


 


1


4


-


1


1



1


1


5


2


-


1


-



-


1


7


2


-


-


-


-



Median precoital time



3



4



3



3



Mean precoital time



3.3



4.3



3.3



3.1



N



10



10



10



10



Table 8. Fertility parameters









































Days of Parameters



Group 1


mg/kg


 


 



Group 2


mg/kg


 


 



Group 3


mg/kg


 


 



Group 4


mg/kg


 



Mating (%)



100



100



100



100



Fertility index (%)



80



80



100



100



Conception index (%)



80



80



100



1000



Gestation index – Breeding (%)



100



100



100



100



Percentage mating = (Females mated / Females paired) * 100
Fertility index = (Females achieving a pregnancy / Females paired) * 100
Conception rate = (Females achieving a pregnancy / Females mated) * 100
Gestation index = (Number of females with living pups / Number of females pregnant) * 100


Table 9. Summary of mean breading data per group.












































































Parameters



Group 1


mg/kg


 


 



Group 2


mg/kg


 


 



Group 3


mg/kg


 


 



Group 4


mg/kg


 



Total Litters


Mean duration of gestation



8


 


21.4



8


 


21.5



10


 


21.7



10


 


21.5



Mean implantation



 


13.3



 


13.5



 


14.0



 


13.3



Post implantation loss



1.4



0.8



1.4



0.5



Dead pups at first litter check



0.3



0.3



0.0



0.0



Live pups at first check



11.9



12.8



12.6



12.8



Postnatal loss days 0- 4



0.1



0.0



0.0



0.0



Live pups day 4



11.8



12.8



12.6



12.8



Birth index (#)



89.6



94.4



90.0



96.2#



Viability index (#)



98.9



100



100



100



# / ## : Fisher's Exact Test significant at 5% (#) or 1% (##) level


Birth index = (number of pups born alive / number of implantations) * 100


Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) * 100


Table 9. summary of organ/bosy weight ratio and percentage mean values.
















































Organs



Group 1 (0


mg/kg bw/day


 


 



Group 2 (100


mg/kg bw/day


 


 



Group 3 (300


mg/kg b/day


 


 



Group 4 (1000


mg/kg bw/day)


 



Body weight


 


 


 


%



366.7


 


 


 


 


 


14.4



357.3


 


 


 


 


 


20.6



373.8


 


 


 


 


 


18.6



360.8


 


 


 


 


 


13.5



Testis (L)


 


 


 


%



1.78


 


 


 


 


0.49



1.72


 


 


 


 


0.48



2.00


 


 


 


 


0.54



1.92


 


 


 


 


0.53



Testis (R)


 


 


 


%



1.75


 


 


 


 


0.48



1.67


 


 


 


 


0.47



1.92


 


 


 


 


0.52



1.92


 


 


 


 


0.53



Epididi (R)


 


 


 


%



0.681


 


 


 


 


 


0.186



0.642


 


 


 


 


 


0.180



0.730


 


 


 


 


 


0.195


 



0.714


 


 


 


 


 


 


0.198



Epidid (L)


 


 


 


 


%



0.675


 


 


 


 


 


0.185



0.644


 


 


 


 


 


0.180



0.743


 


 


 


 


 


0.199



0.738


 


 


 


 


 


0.205



 

Applicant's summary and conclusion

Conclusions:
As no effects were observed, the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.
Executive summary:

In this study, an OECD 421 "“Reproduction /Developmental Toxicity Screening Test” performed to GLP was conducted to generate preliminary information concerning the effects of the test item, Sodium Sulphate, on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.
Four groups (10 males & 10 females) were treated (by gavage) with Sodium Sulphate once daily. Group 1 was the purified water control, group 2, 3 and 4 were dosed at 100, 300 and 1000 mg/kg bw/day, respectively. All were dosed at a dose volume of 10 mL/kg bw. 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 pre-pairing, pairing, gestation and lactation period up to day 4 post partum. 
In parental animals (P0), all animals survived until the scheduled necropsy and no clinical signs were observed. Mean food consumption was not affected by treatment in either sex. No test item-related effects on mean body weight and mean body weight gain were observeed in either sex. Mating performance, fertility index and conception rate were not affected by treatment. The mean number of corpora lutea, the mean number of implantations per dam, and the post-implantation losses were unaffected by test item treatment. The mean duration of gestation was unaffected by treatment. Mean weight of testes and epididymides were not affected by the treatment. All organs and tissues examined did not reveal any macroscopic or microscopic changes related to treatment.


In F1 pups, the number of live pups at first litter check and the mean litter size was unaffected by test item treatment. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment. Mean pup weights and weight gains were also not considered to be affected.


As no effects were observed, the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.