Sodium dimethyl 5-sulphonatoisophthalate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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

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.

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.

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

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.

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.

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 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.

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.

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.

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.

VIABILITY (OFFSPRING): no effects

CLINICAL SIGNS (OFFSPRING): no effects

BODY WEIGHT (OFFSPRING): no effects

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.

Key result

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a reproduction/developmental toxicity screening study according to OECD guideline 421 and GLP (Toxi-Coop Zrt. (e), 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.

Short description of key information:
Based on an OECD Guideline 421 and GLP study with Sodium dimethyl 5-sulphonatoisophthalate, the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

In an OECD Guideline 421 and GLP study with Sodium dimethyl 
5-sulphonatoisophthalate in rats, the NOAEL for development of offspring was at
 the highest test dose of 1000 mg/kg bw/day. Data form structural analogues do
not indicate an alert regarding developmental toxicity. 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1985-03-25 until 1985-06-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

SIM-Ester (CAS No. 3965-55-7) can be reliably assessed taking into account the results of reproduction/developmental toxicity data on the structurally related compounds isophthalic acid (CAS No. 121-91-5, IPA), dimethyl terephthalate (CAS No. 120-61-6, DMT) and terephthalic acid (CAS No. 100-21-0, TPA). Hydrolysis of SIM-Ester to the monomethylester and finally to 5-sulfoisophthalic acid with subsequent urinary excretion has been identified as the main metabolic and excretory pathway (see toxicokinetic section).
The analogues (IPA, DMT and TPA) are toxicologically extensively investigated and structurally closely related to SIM-Ester and its final metabolite 5 -sulfoisophthalic acid. The substances show a toxicological profile comparable to SIM-Ester, i.e. low acute toxicity (LD50 (rat, oral and dermal) > 2000 mg/kg bw; LC50 (rat, inhalation) > 5 mg/L), no sensitization and no genotoxic/mutagenic potential as well as a similar toxicokinetic behavior (OECD, 2001a, 2001b and 2002, Roth T. et al 2013). These similarities justify the read-across from the structural analogues to SIM-Ester regarding toxicity to reproduction. Dimethyl terephthalate is the closest structural analogue for SIM-Ester as it is also a dimethylester. Thus, available experimental data for embryotoxicity taken from analogue DMT are used to fill data gap embryotoxicity as part of a weight of evidence approach.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

Chemical name: Dimethylterephthalate (DMT)
Physical state: solid (at 20°C)
Water solubility: 0.5 g/L (at 20°C)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals from in house breeding (Hoechst AG) (Hoe:WISKf(SPF71))
- Age at study initiation: about 65-70 days (young virgin females)
- Weight at study initiation: about 193 (+/-10) g
- Housing: standard cages with chipped wood used as bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: n. a.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 23°C
- Humidity (%): relative humidity 44 - 64 %
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 1985-03-25 To: 1985-06-04

Route of administration:

oral: gavage

Vehicle:

other: starch mucilage (potato starch (20g /1L distilled water))

Details on mating procedure:

- Impregnation procedure: mating
- If cohoused: Females in estrus cycle were cohoused with males overnight.
- M/F ratio per cage: n. a.
- Length of cohabitation: Until presence of sperm in vaginal smear. Mated females were housed single.
- Proof of pregnancy: Presence of sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

Gestation day 7 to gestation day 16

Frequency of treatment:

daily

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

21 - 22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Due of low toxicity of DMT a limit test was performed

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: n.a.

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 7, 14, 17, and day 21 of pregnancy

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes
Time schedule for examinations: day 0, 7, 14, 17, and day 21 of pregnancy

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Uterus, Placenta

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Placenta weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Blood sampling:

No. At the time study was performed no blood parameter requested by guideline.

Statistics:

YES: Goodman; Exact FISHER

Historical control data:

Yes: Normal ranges at 1986-08-01 from all previous control groups (95 % Probability). The normal range calculations are based on all studies conducted at HOECHST AG since November 1969. Exceptions:
In parameter corpora lutea a tendency toward higher counts has been observed for a long time. Therefore, studies conducted before August 1973 have not been used for the normal range calculations since May 1981.
In parameter implantations a tendency toward higher counts has been observed for a long time. Therefore, studies conducted before August 1973 have not been used for the normal range calculations since May 1981.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Control group: Red discoloration of urine was observed at one female group at gestation day 21 .
Treatment group: At one female alopecia on rump (gestation day 17-21) and in abdomen area was observed (gestation day 18-21). A second female was found wound with scabbing on right side of neck (gestation day 14-21).

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:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Description (incidence and severity):

Not required at OECD 414, version 1981.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not required at OECD 414, version 1981.

Endocrine findings:

not examined

Description (incidence and severity):

Endocrine findings are not required at OECD 414, version 1981.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Description (incidence and severity):

Not required at OECD 414, version 1981.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed

Key result

Abnormalities:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

AGD measurement was no requirement of OECD guideline 414, version 1981.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

not specified

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the results obtained after daily oral treatment of pregnant rats with DMT at a limit dose level of 1000 mg/kg bw/day during gestation day 7 to 16, DMT has no teratogenic potential.

Executive summary:

The embryotoxic property of Terephthalic acid dimethyl ester was investigated in young pregnant female Wistar rats by oral gavage at a limit dose of 1000 mg/kg bw/day solved in Starch mucilage (vehicle). Females were treated from gestation day 7 until gestation day 16. On gestation day 21 the females were killed and Cesarian section was performed. Dams were examined for gross pathological changes. The uterus from all females were removed and the contents were examined. Uteri were examined for number of implantation sites, resorptions, and the number of live and dead fetuses. The number of corpora lutea was counted on ovary. All fetuses were weighed and examined for external malformations. After fetuses have been sacrificed fetuses were sexed and crown to rump length was measured. Approximately half number of fetuses from each dam was examined for skeletal malformations including the one fetus found dead in uterus of a dam of treatment group. The remaining half was evaluated for visceral malformation.

No mortality and no effects on body weight or food consumption was observed in pregnant females until gestation day 21. No clinical signs on behavior were observed with the exemption of two females of the treatment group which had Alopecia on rump (one female gestation day 17 to 21) or in abdomen area (one female gestation day 18-21). One other female of treatment group had a wound with scrabbing on right side of neck from gestation day 14 to 21. One female of control group had a red discoloration of urine on gestation day 21. All dams had live fetuses with exemption of one control dam which had only two empty implantation sites, which indicate the early death after implantation. No abnormal developmental effects and no abnormal pre- or post-implantation losses were noted. The number of corpora lutea, number of implantations und number of live fetuses of treatment group dams was comparable to control group dams.

The fetuses of treatment group were comparable developed to fetuses of control group. Sex ratio of fetuses from treated dams was balanced and comparable to that of controls. In both the treatment group and the control group, male fetuses predominate slightly. One dam of control group had a twin-placenta at which two live fetuses stuck. Number of resorptions was almost comparable between control and treatment group.

Morphological investigations of fetuses did not show any malformation. Internal examinations of the fetal viscera by free-hand-microdissection technique revealed a range of findings which are considered as minimal anomalies, e.g. hematoma in brain and left adrenal and blood in abdominal cavity. Skeletal examination of the Alizarin S stained fetuses revealed a range of variations, i.e. Creation of a short rib at the 7^th cervical vertebra, creation of a short 13^th rib, creation of a 14^th thoracic vertebra and a short 14^th rib, creation of a short and/or normal length 14^th rib at the 1^st lumbar vertebra. Fragmented 12^th thoracic vertebra and fragmented and/or longitudinally displaced sternebrae are considered as minor anomalies. Described waved and/or thickened ribs are assessed as deformations which are reversible postnatally. These findings were assumed to be not test item related as they were found in the same range of control group fetuses. Exception therefrom were the creation of a short rib at the 7^th cervical vertebra, creation of a short 13^th rib, creation of a 14^th thoracic vertebra and a short 14^th rib and creation of a short and/or normal length 14^th rib at the 1^st lumbar vertebra, which were only found at one or two fetuses and thus were assumed to fall within the spontaneous rate. The weak or non-ossification of one or more head bones, non-ossification of one or more sternebrae and non- ossification of metacarpal 5 could be assumed to be retardations. Due to the frequency of this observation seen in control rats it is not a question of retardation in development but the finding of a normal skeletal ossification at Gestation day 21. Only if the number of affected fetuses would be extremely increased one would indicate it as retardation of ossification. However, the number of affected fetuses of the treatment group varied not from number of fetuses of control group. Thus, no indication of delayed ossification was determined in fetuses from treated rats.

Based on the above-mentioned findings it was concluded that the oral application of Terephthalic acid dimethyl ester at a limit dose of 1000 mg/kg bw/day during gestation day 7 to gestation day 16 did not adversely affect the pregnant rats and did not cause any fetal toxicity or teratogenic effect.

Reference 2

Endpoint:

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 reliable GLP compliant guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421

Deviations:

no

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

Species:

rat

Strain:

Wistar

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.

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

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.

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.

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.

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.

Maternal 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 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 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.


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.

Ovaries and uterine content:

NECROPSY
At necropsy special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded. The uterus with cervix, vagina and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved.

HISTOPATHOLOGY
- Detailed histological examination was performed on the ovaries, vagina and uterus, pituitary of the animals in the control and high dose groups and in non-pregnant females. 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.

Fetal examinations:

Pups were carefully examined for gross (external) abnormalities and euthanized on postnatal day 4. Litter weight (on postnatal days 0 and 4) and mean body weight gain per litter (between postnatal days 0-4) were examined.

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.

Indices:

- Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
- Survival Index of pups on postnatal day 4
- Sex ratio % (on postnatal days 0 and 4).

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
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 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 female animals at 1000, 250 and 62.5 mg/kg bw/day during the study (during pre-mating for dams and non-pregnant females, during gestation and lactation periods for dams).

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


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

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
OFFSPRING
Negative effects of the test item on offspring development (mortality, clinical signs, body weight, gross abnormalities and necropsy findings) were not detected between postnatal days 0 and 4.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other: No adverse effects were observed in offspring

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of the present study Sodium dimethyl 5-sulphonatoisophthalate (SIM-Ester) did not cause toxic changes and did not influence the development of the F1 offspring 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The developmental toxicity of SIM-Ester (CAS no. 3965-55-7) can be reliably assessed taking into account the results of the OECD 421 guideline study (see above, "effects on fertility") and the developmental toxicity data on the structurally related compounds isophthalic acid (CAS no. 121-91-5, IPA), dimethyl terephthalate (CAS no. 120-61-6, DMT) and terephthalic acid (CAS no.100-21-0,TPA) in an analogue approach (see section on “repeated dose toxicity” for the justification of the analogue approach and attachment to IUCLID section 13, WoE assessment report). Hydrolysis of SIM-Ester to the monomethylester and finally to 5-sulfoisophthalic acid with subsequent urinary excretion has been identified as the main metabolic and excretory pathway (see toxicokinetic section). The analogues (IPA, DMT and TPA) are toxicologically extensively investigated and structurally closely related to SIM-Ester and its final metabolite 5 -sulfoisophthalic acid. The substances show a toxicological profile comparable to SIM-Ester.

The OECD SIDS on DMT states the following (OECD, 2001a): “The potential for DMT to induce developmental toxicity in rats has been evaluated following an inhalation exposure to DMT at 1 mg/m³ and after gavage exposure to 1,000 mg/kg bw/d throughout gestation. In addition, inhalation exposures to TPA, the primary metabolite of DMT, at exposure levels up to 10 mg/m³ have been assessed during days 6-15 of gestation in rodents. No abnormal developmental effects and no pre- or post-implantation losses were noted in any study.” Also, for isophthalic acid no evidence of teratogenesis or fetotoxicity was observed in rats exposed to 0, 1, 5, or 10 mg/m³ particulate aerosol on gestation days 6 through 15 (OECD, 2002).

In studies performed according to outlines equivalent/similar to OECD guideline 415 (one generation study) no evidence of reproductive or developmental toxicity primarily related to DMT, TPA or IPA has been found (OECD, 2001a, 2001b and 2002). Offspring toxicity was only observed secondary to the formation of renal crystals or calculi which are the primary toxicity manifested in laboratory animals at high dose levels when the renal solubility of the Ca-salt of the respective acid becomes saturated. The reported NOAELs for offspring toxicity are between 240 and 1219 mg/kg bw/d. Due to the higher water solubility of SIM-Ester (32 g/L) and its metabolites (5-sulfo-IPA: 380 g/L) in comparison to the structural analogues (DMT and TPA: 19 mg/L, IPA: 5.4 g/L) similar effects are only expected at doses above the limit dose of 1000 mg/kg bw/d (see also section on “repeated dose toxicity”).

 

In summary, the structural analogues did not induce developmental toxicity (i.e. teratogenic, embryotoxic or fetotoxic effects) when test designs equivalent/similar to OECD guideline 414 were employed. No effects in offsprings which were primarily related to treatment with the analogue substances were seen in studies performed similar/equivalent to OECD guideline 415. Furthermore, SIM-Ester did not cause any substance related effects in the OECD 421 guideline study. Based on these results it can be reliable estimated that SIM-Ester is not a developmental toxicant. Thus, the performance of an OECD 414 guideline study is scientifically not needed and moreover, should not be done considering animal welfare reasons 

Justification for classification or non-classification

Based on the results obtained from reproduction/developmental testing of SIM-Ester and structural analogues, the test substance is not considered to be subject to classification and labelling for toxicity to reproduction/development according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).