Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
28 Jun 2012 to 21 Mar 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Reaction Mass of 4-sulphophthalic acid and 3-sulphophthalic acid (ratio 3:1)
SOURCE: Sulfophthalic acid sodium salts
3. CATEGORY APPROACH JUSTIFICATION
Sulphophthalic acid sodium and ammonium salts are different organic salts of a common acid, sulphophthalic acid. The salts dissociate rapidly in the medium (water) to the common sulphophthalic acid anion and to their different counter ions. The counter ions ammonium and sodium do not influence the solubility and the toxicity of the category members.
4. DATA MATRIX
See Read Across document attached to CSR
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Reaction Mass of 4-sulphophthalic acid and 3-sulphophthalic acid (ratio 3:1)
SOURCE: Sulfophthalic acid sodium and ammonium salts
3. CATEGORY APPROACH JUSTIFICATION
Sulphophthalic acid sodium and ammonium salts are different organic salts of a common acid, sulphophthalic acid. The salts dissociate rapidly in the medium (water) to the common sulphophthalic acid anion and to their different counter ions. The counter ions ammonium and sodium do not influence the solubility and the toxicity of the category members.
4. DATA MATRIX
See Read Across document attached to CSR
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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
Route of administration:
oral: gavage
Vehicle:
water
Details on oral 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
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
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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
Observations and examinations performed and frequency:
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.

CLINCICAL LABORATORY INVESTIGATIONS:
Blood samples were obtained on the day of the scheduled necropsy from 5 males randomly selected from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
Furthermore, an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). Serum samples were stored at ≤-75 °C. Any samples remaining at finalization of the study report, are
discarded. The assay was performed under the responsibility of R. Draheim at Harlan Laboratories Ltd. (Füllinsdorf) under internal laboratory quality control conditions to ensure reliable test results.

HAEMATOLOGY: Yes
The following hematology parameters were determined:
Complete Blood Cell Count:
Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count (total) ,Differential leukocyte count: Platelet count, Reticulocytes
Coagulation:
Prothrombin time (= Thromboplastin time) and Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
The following clinical biochemistry parameters were determined:
Glucose, Urea, Creatinine, Bilirubin (total),Cholesterol (total), Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein (total), Albumin, Globulin, Albumin/Globulin ratio

NEUROBEHAVIOURAL EXAMINATION: Yes
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 post partum) relevant parameters were performed with five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted
following the daily dose administration. Animals were observed for the following:
• Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
• Hand-held observations: muscle tone, constitution, skin, pupil size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
• Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
• Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
• Measurements / Counts: hind limb / fore limb grip strength, rectal temperature.
Any abnormal findings were recorded and, where appropriate, graded in severity.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and
the total activity over 30 minutes were reported.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
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.

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: Yes
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.
Other examinations:
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)
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.
Clinical signs:
no effects observed
Description (incidence and severity):
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.
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 specified
Details on results:
CLINICAL SIGNS AND MORTALITY
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 AND WEIGHT GAIN
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
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.

HAEMATOLOGY
The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level.
Higher reticulocyte counts were observed in females at 300 mg/kg bw/day, and a lower number of large unstained cells occurred at 100 mg/kg bw/day. In the absence of dose-dependency a compound-related effect can be excluded.

CLINICAL CHEMISTRY
The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females at any dose level.
Slightly higher urea levels at 300 and 1000 mg/kg bw/day, as well as slightly higher potassium levels at a dose level of 1000 mg/kg bw/day in females were in the range of the historical control data.

NEUROBEHAVIOUR
No findings were noted during the functional observational battery in males or females at any dose level.
Mean body temperatures of males at 1000 mg/kg bw/day were slightly statistically significantly higher than in the control rats. Since the value of 38°C was in the range of the historical control data (37.5-38.6°C) this finding was considered to be incidental.
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor. Locomotor activity was not affected by the treatment with the test item in males or females at any dose level.

ORGAN WEIGHTS
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 upperer 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
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
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.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic and local
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Key result
Critical effects observed:
no
Conclusions:
.No test item-related toxicity findings were noted during 28-days of exposure to male rats . Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be >1000 mg/kg body weight/day
Executive summary:

There are no oral repeated dose studies on the reaction mass of 4-sulphophthalic acid and 3-sulphophthalic acid (read across target substance). However, an oral repeated dose study is available for the reaction mass of trisodium 4-sulphonophthalate, trisodium 3-sulphonatophthalate, disodium phthalate, and sulphate (read across source substance) and it was used for read-across purposes

The purpose of this study was to generate preliminary information on the possible health hazardslikely to arise from repeated exposure over a relatively limited period of time.The sulphophthalic acid sodium salt reaction mass (read across source substance)was administered to male rats for at least 28 days and to female rats for 14 days.The following dose levels were applied:0(control group), 100, 300, and 1000mg/kg body weight/day. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual bodyweight was used. Control animals were dosed with the vehicle alone (water).The following results were obtained:1)all animals survived until scheduled necropsy, 2) No clinical signs were noted in males and females at any dose level, 3) No adverse effects on mean food consumption and mean body weights of males and femaleswere observed at any dose level.No test item-related findings were noted during the clinical laboratory investigations. Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be >1000 mg/kg body weight/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
Based on the results of a 14-day range-finding study (D52723, BASF project identification 01R0629/11X455) the dose levels of 100, 300, and 1000 mg/kg bw per day were selected for the present study.

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
Details on species / strain selection:
Rat, RccHanTM : WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
- Breeder: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age (at Start of Treatment): 12 weeks
- Body Weight Range (at Start of Treatment): Males: 287 to 348 g; Females: 188 to 222 g
- Identification: Cage card and individual animal number (ear tattoo). Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
- Randomization: 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.
- Acclimatization: Eight days under test conditions after health examination. Only animals without any visible signs of illness were used for the study
- Husbandry: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside the range occasionally occurred for relative humidity. These transient variations were considered not to have any influence on the study. These data are not reported but retained at Harlan Laboratories Ltd. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
- Diet: Pelleted standard Harlan Teklad 2018C (batch nos. 80/11 and 43/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. R

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q-Water
Details on exposure:
- Dose Volume: 10 mL/kg body weight
- Duration of Acclimatization Period: 8 days
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)). Towards the end of treatment (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.
Duration of treatment / exposure:
Males: Minimum 4 weeks; Females: Approximately 6 weeks
Frequency of treatment:
Once daily (within four hours)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
44 males: 11 per group
44 females: 11 per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day range-finding study (D52723 non GLP, BASF project number 01R0629/11X455)
Positive control:
No

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
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 AND COMPOUND INTAKE (if feeding study):
Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 – 14
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.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Oestrous cyclicity (parental animals):
Ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined. Histological examination of ovaries was carried out on the females that did not give birth
Sperm parameters (parental animals):
Testes, epididymides, prostate, and seminal vesicles 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.
Postmortem examinations (parental animals):
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 (incl. Peyer’s patches)
Stomach (forestomach and glandular stomach)
Liver
Kidneys
Adrenals
Lymph nodes (axillary and mesenteric)
Urinary bladder
Aorta
Heart
Thymus
Thyroids and parathyroids
Trachea and lungs (preserved by inflation
with fixative and then immersion)
Pituitary gland
Spleen
Peripheral nerve (sciatic)
Bone marrow (femur)
Femur with knee joint
Mammary gland (male and female)
Pancreas
Eyes with optic nerve and harderian gland
Lacrimal gland
Larynx
Nasal cavity
Esophagus
Salivary glands – mandibular, sublingual
Skeletal muscle
Sternum with bone marrow
Pharynx
Statistics:
The following statistical methods were used to analyze food consumption, body and organ weights, clinical laboratory and reproduction data, locomotor activity 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:
- Number of females paired
- Number of females mated
- Number of pregnant females
- Number of females which reared their pups until day 4 post partum
Offspring viability indices:
Litter size
Sex ratios
Posnatal loss Days 0-4 Post partum

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
- 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.
Mortality:
no mortality observed
Description (incidence):
There were no test item-related deaths. All animals survived until scheduled necropsy
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- There were no effects on mean body weight gain and mean body weights of male and female rats 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).
- 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 and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- 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 prepairing period a trend was observed. Differences in mean food consumption were not statistically significant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
- The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level.
- Higher reticulocyte counts were observed in females at 300 mg/kg bw/day, and a lower number of large unstained cells occurred at 100 mg/kg bw/day. In the absence of dose-dependency a compound-related effect can be excluded.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females at any dose level.
- Slightly higher urea levels at 300 and 1000 mg/kg bw/day, as well as slightly higher potassium levels at a dose level of 1000 mg/kg bw/day in females were in the range of the historical control data.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No findings were noted during the functional observational battery in males or females at any dose level. Mean body temperatures of males at 1000 mg/kg bw/day were slightly, but statistically significantly higher than in the control rats. Since the value of 38.2°C was in the range of the historical control data (37.5-38.6°C) this finding was considered to be incidental. Locomotor activity was not affected by the treatment with the test item in males or females at any dose level.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- Under the conditions of this experiment, the test item 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- 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.
- 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.
- 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.
- 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.
- No effects on postnatal loss were observed at any dose level.

Reproductive function / performance (P0)

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

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- 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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
- No abnormal findings were noted at first litter check or during the first 4 days post partum.
- No test item-related findings were noted at macroscopic examination of F1 pups
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- 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

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. (total fraction)
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
gross pathology

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on these results, the NOAEL (No Observed Adverse Effect Level) for general and reproductive toxicity was considered to be >1000 mg/kg body weight/day
Executive summary:

This reproductive toxicity screening study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item reaction mass of sulphophthalic acid sodium salt (read across source substance) to rats. The test item was administered in Milli-Qwater as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Neither clinical signs nor adverse effects on mean food consumption, mean body weight, reproduction and breeding or pup development were noted. No test item-related findings were noted during the histological examination. Based on these results the NOAEL (No Observed Adverse Effect Level) for general and reproductive toxicity was considered to be >1000 mg/kg body weight/day.