Reaction mass of 1,2-Benzenedicarboxylic acid, 3-sulfo-, ammonium salt (1:3), 4-Sulphophthalic acid, ammonium salt, Ammonium sulphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Produkt SPS

IUPAC Name:

Produkt SPS

Details on test material:

- Name of test material (as cited in study report): Produkt SPS
- Test-substance No.: 11/0629-1
- Lot/batch No.: 78522224U0
- Purity: Trisodium 4-sulphonophthalate: 25.5 g/100 g; Trisodium 3-sulphonatophthalate: 7.6 g/100 g; Disodium phthalate: 1 g/100 g; Sulphate: 60.6 g/100 g; Water: 4.8 g/100 g; Sum: 99.5 g/100 g; Determined by 1H-NMR-analysis
- Homogeneity: The test substance was homogeneous by visual inspection
- Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage conditions: Room temperature
- Physical state/ colour: Solid/ white
- Expiry date: December 10, 2012

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: 12 weeks
- Weight at study initiation: Males: 287 to 348 g; Females: 188 to 222 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Eight days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test item as supplied by the Sponsor.
Produkt SPS was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance was soluble in the vehicle

Details on mating procedure:

During the pairing period, females were housed with sexually mature males from the same dose group (1:1) until evidence of copulation was observed. The females were removed and housed individually when:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.
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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group (middle only) as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were
taken from the middle to confirm the stability (8 days at room temperature (20 ± 5 °C)). On 24- Aug-2012, samples were taken from the middle to confirm the concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. B. Ludwig
(Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were discarded after approval of the data by the
sponsor.
Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were discarded after approval of the data.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 6 weeks

Frequency of treatment:

Once daily

Details on study schedule:

N/A

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on 14-day range finder.
- Rationale for animal assignment (if not random): Performed on the fifth day of acclimatization using a computer-generated random algorithm. Body
weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Positive control:

N/A

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality, Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Detailed clinical observations:
Once prior to the first administration of the test item (day 6 of acclimatization) and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena. Animals were observed
for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or
tonic movements, stereotypies or bizarre behavior were also reported.

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

FOOD CONSUMPTION:
Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 and weekly after pairing period.
Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 - 14 and 14 – 21 and days 1 - 4 of the lactation.
No food consumption was recorded during the pairing period.

Oestrous cyclicity (parental animals):

N/A

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:
Organ weights:
Uterus (including cervix), Prostate, Ovaries (weighed as pairs), Seminal vesicles (inclusive coagulating gland)
Histopathology
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on the females that did not give birth.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed
individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Pups were sacrificed on day 4 post partum. Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Postmortem examinations (parental animals):

SACRIFICE
Males were sacrificed after treatment of 28 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum.
Since in several females a birth did not occur on the expected date (day 21 post coitum), the dams were sacrificed on day 25 post coitum.

GROSS NECROPSY
All animals sacrificed were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
Dead pups, except those excessively cannibalized, were examined macroscopically.
All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of apparently non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS

TISSUE PRESERVATION:
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative)
The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Ovaries (with oviduct), Uterus (with vagina)
In addition, from all males and females the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions, Brain, Spinal chord (cervical, thoracic, lumbar), Small and large intestines[2] (incl. Peyer’s patches), Stomach (forestomach and glandular stomach), Liver, Kidneys, Adrenals, Lymph nodes (axillary and mesenteric), Urinary bladder, Aorta[1], Heart, Thymus, Thyroids and parathyroids, Trachea and lungs (preserved by inflation with fixative and then immersion), Pituitary gland[1], Spleen, Peripheral nerve (sciatic), Bone marrow (femur), Femur with knee joint[1], Mammary gland (male and female)[1], Pancreas[1], Eyes with optic nerve and harderian gland[1], Lacrimal gland[1], Larynx[1], Nasal cavity[1], Esophagus[1], Salivary glands – mandibular, sublingual[1], Skeletal muscle[1], Sternum with bone marrow[1], Pharynx[1]
[1] only examined by histopathology in case of macroscopic findings indicative of potential toxicity
[2] duodenum, jejunum, ileum, colon, caecum, rectum

HISTOPATHOLOGY:
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined. The same applied to all occurring gross lesions. The remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Histological examination of ovaries was carried out on the females that did not give birth.

ORGAN WEIGHTS:
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.
In addition, from 5 males and 5 females killed at the end of the study which were selected for hematology and clinical chemistry examination from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Uterus (including cervix), Prostate, Liver, Thymus, Spleen, Thyroid (after fixation), Ovaries (weighed as pairs), Seminal vesicles (inclusive coagulating gland)

Postmortem examinations (offspring):

SACRIFICE
Pups were sacrificed on day 4 post partum.

GROSS NECROPSY
Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:

The following statistical methods were used to analyze food consumption, body and organ weights, clinical laboratory and reproduction data and macroscopical findings:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

fertility indices, mean precoital time, post-implantation losses (absolute and percent)

Offspring viability indices:

mean litter size, pup sex ratios and viability indices.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test item-related deaths. All animals survived until scheduled necropsy. No clinical signs were noted in males and females at any dose level. No findings at detailed weekly clinical observation were noted in males and females at any dose level.

BODY WEIGHT(PARENTAL ANIMALS)
MALES:
Pre-pairing and Pairing Periods
There were no effects on mean body weight gain and mean body weights at any dose level and in any study phase.
The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +15%, +14%, +13% and +14% during the pre-pairing period and +5%, +5%, +5% and +6% during the pairing period (percentages refer to the body weight gain within the period).
FEMALES:
Pre-pairing, Pairing, Gestation and Lactation Periods
No test item-related effects on mean body weight and mean body weight gain were noted. At a dose level of 1000 mg/kg bw/day, statistically significantly higher mean body weights were noted (+6% compared to the control group) at the end of the study. Higher (but not statistically significantly different) mean body weight gain was already noted during the pre-pairing period. This increase was accompanied by higher mean food consumption during the gestation and lactation period. However, in the absence of any other compound-related effect, this minor difference is considered to be rather fortuitous than compound-related.
The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +8%, +8%, +7% and +9% during the pre-pairing period, +55%, +49%, +54% and +58% during the gestation period and +7%, +6%, +9% and +5% during the lactation period (percentages refer to the body weight gain within the period).

FOOD CONSUMPTION (PARENTAL ANIMALS)
MALES:
Pre-Pairing Period
There were no effects on mean food consumption at any dose level and in any study phase.
FEMALES:
Pre-pairing, Gestation and Lactation Periods
There were no effects on mean food consumption at any dose level and in any study phase. At 1000 mg/kg bw /day, during the gestation and lactation period, mean food consumption was slightly higher (+10% and +15%, respectively) compared to the control group. Also in the pre-pairing
period a trend was observed. Differences in mean food consumption were not statistically significant.


Mating Performance and Fertility
No effects on mating performance and fertility were observed at any dose level.
Two females (one control animals and one treated at 100 mg/kg bw/day) were mated within the second pairing period. All other females were mated within the first pairing period. For female no 47 in the control group, mating was neither detected during the first nor the second pairing
period. However, mating during the first pairing period proved to have been successful.
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 2.6, 2.4, 3.6 and 3.1 days in with ascending dose levels. The median precoital time was 3 days in controls and at 300 and 1000 mg/kg bw/day, and 2 days at 100
mg/kg bw/day.
Two females each from the control group and at 300 mg/kg bw/day, and one female treated at 100 mg/kg bw/day were not pregnant. As a result the fertility index in controls and at 300 mg/kg bw/day was 81.8%, at 100 mg/kg bw/day it was 90.9% and at 1000 mg/kg bw/day it was 100.0%.

Corpora Lutea Count
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (15.3, 13.2, 16.0 and 15.1 in order of ascending dose level) and gave no indication of a test item-related effect.

Duration of Gestation
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.4, 21.5, 21.4 and 21.5 days, in order of ascending dose level.

Implantation Rate and Post-implantation Loss
No effects on implantation rate or post-implantation loss were observed at any dose level. The mean number of implantations per dam was 12.9, 11.0, 13.6 and 13.0 in order of ascending dose levels. The mean incidence of post-implantation loss as a percentage of total implantations was 6.0%, 9.1%, 7.4% and 7.7%, in order of ascending dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males and females no test item-related effects on organ weights were noted.
Absolute testis weights in males at 1000 mg/kg bw/day were statistically significantly higher, confirmed by the values relative to body weight. At this dose level, the testes weight of one male (no. 41) was abnormally high and microscopically a minimal sperm arrest (isolated finding) was observed for this male. Also at 300 mg/kg bw/day, higher absolute (not statistically significant) and relative to body weight (statistically significant) testes weights were noted. All values were in the range of the historical control data however controls at the lower limit and animals treated at 300 and 1000 mg/kg bw/day at the upper limit. In general, no microscopical correlation to the higher testes weights was noted. Due to these facts, the higher testes weights were considered to be a result of biological variability and not a test item-related effect.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test item-related findings noted at necropsy in males and females.
All gross lesions recorded were considered to be within the range of normal background alterations and showed no dose dependency.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Under the conditions of this experiment, the test item “Produkt SPS” did not induce any test item-related findings in reproductive organs or other organs examined.
The qualitative sperm staging did not reveal any relevant differences in stages of spermatogenesis between control males and males at 1000 mg/kg bw/day. All the findings in reproductive organs were considered to be within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

NOAEL

Remarks:

for Reproduction and Developmental Toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Results: F1 generation

Details on results (F1)

LITTER SIZE AT FIRST LITTER CHECK
No effects on litter size were observed at any dose level.
Mean litter size at first litter check was 12.1, 10.0, 12.6 and 12.1 pups in order of ascending dose levels. No dead pups at first litter check were recorded in any group.

POSTNATAL LOSS DAY 0 - 4 POST PARTUM
No effects on postnatal loss were observed at any dose level.

LITTER DATA - F1 PUPS
External Examination at First Litter Check and during Lactation:
No abnormal findings were noted at first litter check or during the first 4 days post partum.

Sex Ratios
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.
The proportion of males at first litter check was 49, 52, 47 and 43%, in order of ascending dose level.

Body Weights to Day 4 Post Partum
Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item.
On day 1 post partum mean pup weights were 5.9, 5.9, 6.0 and 6.4 g in order of ascending dose level. Also mean body weight gain was similar in all groups on day 4 post partum and no effects on mean body weights were recorded.

Macroscopical Findings
No test item-related findings were noted at macroscopic examination of F1 pups.

Overall reproductive toxicity

Reproductive effects observed:

not specified