Sodium dimethyl 5-sulphonatoisophthalate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-19 to 2012-11-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reliabel GLP compliant guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 "Reproduction/Developmental Toxicity Screening Test", EPA Health Effects Test Guidelines, July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium dimethyl 5-sulphonatoisophthalate

EC Number:

223-578-8

EC Name:

Sodium dimethyl 5-sulphonatoisophthalate

Cas Number:

3965-55-7

Molecular formula:

C10H10O7S.Na

IUPAC Name:

sodium 3,5-bis(methoxycarbonyl)benzenesulfonate

Test material form:

other: solid

Details on test material:

- Name of test material: Sodium dimethyl 5-sulphonatoisophthalate (SIM-Ester)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation: parental male and female animals: 85 – 90 days old,
- Weight at study initiation: (P) Males: 315 – 376 g; Females: 199 – 245 g´
- Housing:
Before mating: 2 animals of the same sex/ cage
Mating hours: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: 2 animals / cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Air changes: 8 – 12 air exchanges/hour by central air-condition system
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility for not longer than three days.

VEHICLE
- Justification for use and choice of vehicle: The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability in the chosen vehicle was verified. SIM-Ester was stable for 4 hours at room temperature (recovery: 99 % and 108 % of nominal concentrations of ca. 1 mg/mL and ca. 200 mg/mL, respectively) and for three days in refrigerator (recovery: 98 % and 104 % of nominal concentrations of ca. 1 mg/mL and ca. 200 mg/mL, respectively).
- Concentration in vehicle: 12.5, 50, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL dose preparation/kg body weight
- Lot/batch no. : N83746634

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 1-4 days
- Proof of pregnancy: Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421).
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing solutions (control of concentration and homogeneity) was performed. Five samples were taken from different places from each concentration on both occasions and 1x5 mL sample were taken from the control solution (group 1) and analyzed. Concentration of the test item in the dosing solutions varied in the range of 97 and 109 % of the nominal values at both analytical occasions.

Duration of treatment / exposure:

Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Male animals were dosed for 41 days and then they were subjected to necropsy one day after the last treatment. Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 - 5 [for 41 to 44 days, depending on day of mating (mating days 1-4)]. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant female animals were treated up to and including the day before necropsy (for 41 days).

Frequency of treatment:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 male and 12 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting based on the available toxicity data indicating no/low oral toxicity.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, after treatment at approximately the same time. More detailed examinations were made weekly prior to and during the mating until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling.

BODY WEIGHT: Yes
- Time schedule for examinations: Parent male animals were weighed on the first day of dosing (day 0), weekly thereafter and on the day of necropsy.
Parent females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partal days 0 (within 24 hours after parturition) and 4, as well as on the day of necropsy. Female animals were weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.


FOOD CONSUMPTION:
- The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period except mating days (pre-mating and post mating for male animals and non-pregnant females, during pre-mating, gestation days 0, 7, 14 and 21, lactation days 0 and 4 for dams).

EXAMINATION OF PLACENTAL SIGN:
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on day 13 of gestation. If the test was negative on day 13 the examination was repeated on day 14 of gestation.

OBSERVATION OF THE DELIVERY PROCESS
- Females were allowed to litter and rear their offspring. Delivery process was monitored whilst keeping possible interferences at a minimum. Observations were reported individually for each animal. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and cover their newborns or not. The sucking success was monitored by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.

Sperm parameters (parental animals):

Parameters examined in all male parental animals of the 1000 mg/kg bw/d and control group:
testis weight, epididymis weight, quantity and morphologically of the various spermatogenic cells (spermatogonia, spermatocytes, spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities. Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete) and on day 4 post-partum with an accuracy of 0.1 g. In addition to the observations on parent animals, any abnormal behavior of the offspring was monitored. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead were subjected to necropsy by a macroscopic examination. On day 0 birth, a lung flotation test was performed on all dead pups to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.

Postmortem examinations (parental animals):

GROSS NECROPSY
- Gross necropsy was performed on each animal one day after the last treatment (day of sacrificing). Animals were anesthetized by Isofluran and then were exsanguinated. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded. The testes, epididymides and brain of all male adult animals were weighed. The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were fixed in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.


HISTOPATHOLOGY
- Detailed histological examination was performed on the ovaries, vagina and uterus, pituitary, testes and epididymides of the animals in the control and high dose groups and in non-pregnant females and males cohabited with at 62.5 mg/kg bw/day. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

Necropsy of dead pups was performed.

Statistics:

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
The frequency of clinical signs, pathology and histopathology findings were calculated. Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value.

Reproductive indices:

Mating Index
Fertility Index
Gestation Index

Offspring viability indices:

Survival Index of pups on postnatal day 4 Survival Index of pups on postnatal day 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods) in each group. Grayish color of the stool was observed in all cages of animals (male and female) at 1000 and 250 mg/kg bw/day from Days 2 and 4, respectively, up to the end of the observation period.

Mortality:

no mortality observed

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals.

Details on results (P0)

MORTALITY
There was no test item related mortality at any dose level (1000, 250 and 62.5 mg/kg bw/day).

CLINICAL OBSERVATION
The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods) in each group. Grayish color of the stool was observed in all cages of animals (male and female) at 1000 and 250 mg/kg bw/day from Days 2 and 4, respectively, up to the end of the observation period.

BODY WEIGHT AND BODY WEIGHT GAIN
The body weight development was undisturbed in the test item treated animals at each dose level (1000, 250 and 62.5 mg/kg bw/day) during the entire treatment period (pre-mating, mating, post-mating, gestation and lactation periods).

FOOD CONSUMPTION
The mean daily food consumption was not affected by the test item in male or female animals at 1000, 250 and 62.5 mg/kg bw/day during the study (pre-mating and post mating for male animals, during pre-mating for dams and non-pregnant females, during gestation and lactation periods for dams).

REPRODUCTION
There were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals.

NECROPSY
Specific macroscopic alterations related to the test item were not found during the necropsy.

ORGAN WEIGHT
There were no test item related changes in testes and epididymides weights.

HISTOPATHOLOGY
Histopathological examinations of male and female genital organs (ovaries, uterus, cervix vagina, testes and epididymides) and pituitary did not reveal any toxic or other test item related changes at any dose level.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related effects were noted.

2

Key result

Dose descriptor:

NOAEL

Remarks:

(reproductive performance)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related effects were noted.

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING): no effects

CLINICAL SIGNS (OFFSPRING): no effects

BODY WEIGHT (OFFSPRING): no effects

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related effects were noted.

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study Sodium dimethyl 5-sulphonatoisophthalate (SIM-Ester) did not cause toxic changes and did not influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats from conception to day 4 post-partum after repeated dose oral administration at 1000, 250 or 62.5 mg/kg bw/day.

Executive summary:

In a reproduction/developmental toxicity screening according to OECD guideline 421 and GLP (Toxi-Coop Zrt., 2012) Sodium dimethyl 5 -sulphonatoisophthalate (SIM-Ester) was administered by gavage to groups of 12 rats per sex and dose at 0, 62.5, 250 and 1000 mg/kg bw/d. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy (altogether for 41 days). For females, test item was administered through the gestation period and up to lactation days 3-5 for 41-14 days, i.e. up to the day before the necropsy (altogether for 41-44 days). The test item was administered in 0.5% Methylcellulose as vehicle at a dose volume of 5 mL/kg bw/day. Control animals were doses with the vehicle alone. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process. The dams were allowed to litter, and rear their young up to day 4 postpartum. All parental animals were subjected to gross pathology one day after the last treatment. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups. The reproductive organs and pituitary of non-pregnant females and males cohabited with in the low dose group were also processed and evaluated histologically. Pubs were examined for sex distribution, survival, clincial sings, gross abnormalities and body weight. Necropsy on dead pups was performed.

The only effect seen in parental animals was a grayish color of the stool in all cages of animals (male and female) at 1000 and 250 mg/kg bw/day from Days 2 and 4, respectively, up to the end of the observation period. Based on this finding a NOAEL of 1000 mg/kg bw/d was derived for general toxicity as well as for reproductive toxicity.

Negative effects of the test item on offspring development (mortality, clinical signs, gross abnormalities, body weight and necropsy findings in dead pups) were not detected between postnatal days 0 and 4. Thus, the NOAEL fo 1000 mg/kg bw/d was established for the F1 generation.