Sodium hydrogen-5-sulphoisophthalate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 2012 to 27 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 2012 to 27 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CrI:WI(Han) (outbred, SPF-Quality)

Details on test animals or test system and environmental conditions:

Refer to Section 7.5.1 of this IUCLID file

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Refer to Section 7.5.1 of this IUCLID file

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Refer to Section 7.5.1 of this IUCLID file

Details on mating procedure:

Refer to Section 7.5.1 and 7.8.1 of this IUCLID file

Duration of treatment / exposure:

Refer to Section 7.5.1 and 7.8.1 of this IUCLID file

Frequency of treatment:

Refer to Section 7.5.1 and 7.8.1 of this IUCLID file

Duration of test:

Refer to Section 7.5.1 and 7.8.1 of this IUCLID file

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Refer to Section 7.5.1 and 7.8.1 of this IUCLID file

Maternal examinations:

Refer to Section 7.5.1 and 7.8.1 of this IUCLID file

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

Not applicable

Statistics:

Refer to Section 7.5.1 and 7.8.1 of this IUCLID file

Indices:

Liters (total)
Duration of gestation
Dead pups at first litter check
Living pups at first litter check
Postnatal loss
Viability index (%)

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: Refer to Section 7.5.1 of this IUCLID file

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Three pups of the control group and one pup at 300 mg/kg were found dead on Day 1 or missing on Day 2 of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Changes in sex ratio:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, sex ratio of the pups indicated more female than male pups (62% female pups compared to 47% of the control group); this was statistically significantly different from controls. The laboratory’s historical control data (97 screening studies, years 2008-2012) includes two studies in which a comparable percentage of female pups was noted. As these two historical studies show the ultimate limit of the historical control data range, a possible relationship to treatment could not be excluded for the changed sex ratio in the current study. This was strengthened by the fact that seven out of nine litters in this group showed more female than male pups. The change in sex ratio may be indicative of a decrease in anogenital distance for the male pups leading to a ‘misclassification’ into female pups, rather than a direct effect on sex ratio (sexing was done using anogenital distance).

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

effects observed, treatment-related

Description (incidence and severity):

See remark under "changes in sex ratio". The change in sex ratio may be indicative of a decrease in anogenital distance for the male pups leading to a ‘misclassification’ into female pups, rather than a direct effect on sex ratio.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

At macroscopic examination, no milk in the stomach was noted for three litters of the control group, one litter at 100 mg/kg bw/day and four litters (not all pups) at 300 mg/kg. This was not considered to be treatment related or toxicologically relevant since no dose response relationship was noted, it also occurred in the control group, and these pups survived until the scheduled necropsy, indicating they were all sufficiently fed during lactation. This finding for the animals surviving to the scheduled necropsy is likely secondary to the time when the actual necropsies were performed, and is not attributable to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: anogenital distance

Description (incidence and severity):

A change in sex ratio was observed, which may be indicative of a decrease in anogenital distance for the male pups leading to a ‘misclassification’ into female pups (rather than a direct effect on sex ratio).

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

yes

Developmental data summary

	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Litters
Total	9	9	10	9
Dration of gestation
Mean	21.3	21.1	21.3	21.3
St Dev	0.5	0.6	0.5	0.5
N	9	9	10	9
Dead pups at first litter check
Litters affected (%)	1	0	0	0
Total	1	0	0	0
Mean	0.1	0	0	0
St Dev	0.3	0.0	0.0	0.0
N	9	9	10	9
Living pups at first litter check
% males/%females	53/47	53/47	54/46	38/62#
Total	93	104	92	102
Mean	10.3	11.6	9.2	11.3
St Dev	2.5	3.2	3.7	2.9
N	9	9	10	9
Postnatal loss
% of living pups	2.2	0.0	1.1	0.0
Litters affected (No.)	1	0	1	0
Total (No.)	2	0	1	0
Mean	0.2	0.0	0.1	0.0
St Dev	0.7	0.0	0.3	0.0
N	9	9	10	9
Viability Index (No.)	97.8	100.0	98.9	100.0

# Fischer's exact test significance at 5%

Conclusions:

A screening study for development and reproduction toxicity was performed according to OECD guideline 422 and GLP principles. Developmental toxicity was observed at 1000 mg/kg bw/day as a possible relationship to treatment could not be excluded for the change in sex ratio. The NOAEL for developmental effects was 300 mg/kg bw/day.

Executive summary:

A screening study for development and reproduction toxicity was performed according to OECD guideline 422 and GLP principles. At 1000 mg/kg, sex ratio of the pups indicated more female than male pups (62% female pups compared to 47% of the control group); this was statistically significantly different from controls. The laboratory’s historical control data (97 screening studies, years 2008-2012) includes two studies in which a comparable percentage of female pups was noted. As these two historical studies show the ultimate limit of the historical control data range, a possible relationship to treatment could not be excluded for the changed sex ratio in the current study. This was strengthened by the fact that seven out of nine litters in this group showed more female than male pups.

It should be noted that this change in sex ratio may be indicative of a decrease in anogenital distance for the male pups leading to a ‘misclassification’ into female pups, rather than a direct effect on sex ratio.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 2012 to 27 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to an internationally recognized protocol and all relevant GLP requirements were fulfilled.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: US EPA OPPTS 870.3650, July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CrI:WI(Han) (outbred, SPF-Quality

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 11 weeks
- Weight at study initiation: males, 309-317 g (day 0, mean); females, 200-203 g (day 0, mean)
- Fasting period before study: none
- Housing:
Pre-mating: animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: females were caged together with males (one-to-one).
Post-mating: males housed in home cages and females housed individually in Macrolon plastic cages.
Pups: kept with dams until termination. During locomotor monitoring of dams pups were kept warm in home cages with bottles filled with warm water. Pups were not kept from dams for more than 30-40 minutes.
Locomotor Activity: Animals housed in Hi-temp polycarbonate cages (Anacare corp., USA; 48.3 x 26.7 x 20.3 cm).
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF(R) Spezialdiaten GmbH, Soest,Germany)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days prior to the start of treatments

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): approx. 15/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 August 2012 To: 27 September 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.

Doses were administered by plastic feeding tube.

Dose volume: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (11 September 2012), according to a validated method (Project 500503). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 41, 43 (Group 1), 62 , 70 (Group 3) and 77 (group 4) were not dosed during littering.

Frequency of treatment:

Once daily, 7 d/week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose levels

Based on the results of a 10-day dose range finding study (Project 500603), the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.

Study outline

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43 - 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Positive control:

Not applicable

Observations and examinations performed and frequency:

The following observations and examinations were evaluated (see Sections 7.8.1 and 7.8.2 of this IUCLID for details on reproductive and developmental effects):

mortality / viability, clinical signs (daily)
functional observations and locomotor activity (end of treatment)
body weight and food consumption (at least at weekly intervals)
clinical pathology (end of treatment)
macroscopy at termination
organ weights and histopathology on a selection of tissues

Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

Clinical laboratory investigations

Blood samples were collected from the selected 5 animals/sex/group (see Allocation) under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.

The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids. The following parameters were determined:

Parameter Abbreviation Unit

Haematology
White blood cells WBC 10^9/L
Differential leucocyte count %WBC
neutrophils, lymphocytes, monocytes, eosinophils, basophils
Red blood cells 10^12/L
Reticulocytes %RBC
Red blood cell distribution width RDW %
Haemoglobin mmol/L
Haematocrit L/L
Mean corpuscular volume MCV fl
Mean corpuscular haemoglobin MCH fmol
Mean corpuscular haemoglobin concentration MCHC mmol/L
Platelets 10^9/L

Clotting Potential
Prothrombin time PT s
Activated Partial thromboplastin time APTT s

Clinical Biochemistry
Alanine aminotransferase ALAT U/L
Aspartate aminotransferase ASAT U/L
Alkaline phosphatase ALP U/L
Total Protein g/L
Albumin g/L
Total Bilirubin μmol/L
Urea mmol/L
Creatinine μmol/L
Glucose mmol/L
Cholesterol mmol/L
Sodium mmol/L
Potassium mmol/L
Chloride mmol/L
Calcium mmol/L
Inorganic Phosphate Inorg. Phos mmol/L
Bile acids μmol/L

Sacrifice and pathology:

All males and selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.

Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days:

Condition Day of necropsy

Females which delivered Lactation Days 5-7.
Females which failed to deliver (nos. 50, 52 and 78) Post-coitum Day 27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Males Following completion of the mating period (a minimum of 28 days of dose
administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

In selected 5 animals/sex/group:
Identification marks: not processed; Ovaries; Adrenal glands; (Pancreas); (Aorta); Peyer's patches [jejunum, ileum] if detectable; Brain - cerebellum, mid-brain, cortex; Pituitary gland; Caecum; Preputial gland; Cervix; Prostate gland; Clitoral gland; Rectum; Colon; (Salivary glands - mandibular, sublingual); Coagulation gland; Sciatic nerve; Duodenum; Seminal vesicles; Epididymides (1); Skeletal muscle; Eyes (with optic nerve (if detectable) and Harderian gland) (1); (Skin); Spinal cord -cervical, midthoracic, lumbar; Female mammary gland area; Spleen; Femur including joint; Sternum with bone marrow; Heart; Stomach; Ileum; Testes (1); Jejunum; Thymus; Kidneys; Thyroid including parathyroid if detectable; (Lacrimal gland, exorbital); (Tongue); (Larynx); Trachea; Liver; Urinary bladder; Lung, infused with formalin; Uterus; Lymph nodes - mandibular, mesenteric; Vagina; (Nasopharynx); All gross lesions; (Esophagus)

In all remaining animals, females failing to deliver (2):
Cervix; Prostate gland; Clitoral gland; Seminal vesicles; Coagulation gland; Testes (1); Epididymides (1); Uterus; Ovaries; Vagina; Preputial gland; All gross lesions; Identification marks: not processed

(1) Note: Fixed in modified Davidson's solution (prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
(2) Note: In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Tissues/organs indicated in parentheses (above) were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Histopathology

All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

From a selected 5 males from the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

A peer review on the histopathology data was performed by a second pathologist. The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4 (see Allocation).
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 (see Allocation) and all males suspected to be infertile, to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- Stomach and kidneys of all selected 5 animals (see Allocation) of Groups 2 and 3 based on (possible) treatment-related changes in these organs in Group 4.
- Epididymides of all selected 5 males (see Allocation) of Groups 2 and 3 based on (possible) treatment-related changes in this organ in Group 4.
- The reproductive organs* of males 10 (Group 1), 12 (Group 2) and 38 (Group 4) that failed to sire and females 50 (Group 1; not pregnant), 52 (Group 2; not mated) and 78 (Group 4; not mated) that failed to deliver healthy pups:

* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

All abnormalities were described and included in the report. An attempt was made to correlate gross
observations with microscopic findings.

Other examinations:

Organ Weights
The following organ weights and terminal body weights were recorded from selected 5 animals/sex/group on the scheduled day of necropsy: Adrenal glands; Spleen; Brain; Testes; Epididymides; Thymus Heart; Uterus (including cervix); Kidneys; Prostate (weighed when fixed at least 24 h); Liver; Seminal vesicles including coagulating glands; Ovaries; Thyroid including parathyroid.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

No mortality occurred during the study period.

Salivation seen after dosing among animals of the 1000 mg/kg dose group for only 1 to 3 days was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Incidental findings that were noted included scabs, a wound and alopecia. As this was observed for two control animals, it was not treatment related.

BODY WEIGHT AND WEIGHT GAIN

Body weights and weight gains of treated animals remained in the same range as controls.

FOOD CONSUMPTION

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted. Slightly increased values for relative food consumption noted for high dose females during postcoitum Days 0-4 and 14-17 were not considered toxicologically relevant as the increases were slight,
values were within normal limits, and were not consistent over time.

HAEMATOLOGY

Haematological parameters of treated rats were considered not to have been affected by treatment. The statistically significantly decreased value for percentage of eosinophils for high dose males was not considered toxicologically relevant, as the decrease was slight and the value was within normal limits.

CLINICAL CHEMISTRY

At 1000 mg/kg, several changes were noted for clinical biochemistry parameters. Decreased values for total protein (both sexes), albumin (males) and creatinine (females) were noted for high dose animals. In addition, glucose concentration was increased for all treated males (statistically significant for low and high dose), but without a clear dose response relationship. See tabular data below.

The remaining statistically significant changes were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution, remained within the range considered normal for rats of this age and strain and/or were considered due to slightly high control values.

NEUROBEHAVIOUR

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.

The variation in motor activity did not indicate a relation with treatment. The statistically significantly increase noted for high dose females were considered due to very low control values. Historical control data (N=211; 39 studies; years 2010-2012) show for females at the end of lactation a mean value for total movements of 4441 (P5 2006 and P95 7604) and for ambulations 1060 (P5 408 and P95 2100).

All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS

No toxicologically relevant changes were noted in organ weights and organ to body weight ratios up to 1000 mg/kg.

The statistically significantly increased kidneys weights noted for high dose females were considered not to be a sign of toxicity as the values were within normal limits (with the controls at the lower end). Moreover, no microscopic abnormalities were noted for the female kidneys.

GROSS PATHOLOGY

Nodules (yellowish, soft) were recorded in the epididymides for males of all groups, with an incidence of two in the control group, one at 100 mg/kg, one at 300 mg/kg and three at 1000 mg/kg. In addition, one low dose male showed a greenish, soft nodule in the perirenal adipose tissue.

The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included pelvic dilation, foci at the stomach, thymus or clitoral glands, scab formation, dark red discolouration of the mandibular lymph nodes, diaphragmatic hernia of the liver, alopecia, nodule in the kidneys, and enlarged spleen.

HISTOPATHOLOGY: NON-NEOPLASTIC

Microscopic findings were noted at 1000 mg/kg in the epididymides, kidneys and stomach.

Sperm granuloma were seen in the epididymides at moderate degree in two Group 1 (controls), one Group 2 (bilateral - slight and moderate) (100 mg/kg), in one Group 3 (bilateral - slight and severe) (300 mg/kg) and at minimal or moderate degree (two instances were bilateral) in five Group 4 (1000 mg/kg) males. This alteration was not statistically significant in any group when compared to controls. This finding was the microscopic correlate to the nodules noted in this organ at necropsy.

In the kidneys, hyaline droplets in cortical tubules were noted at minimal or slight degree in three Group 1 (controls), minimal in four Group 2 (100 mg/kg), minimal or slight in three Group 3 (300 mg/kg) and at minimal to moderate degree in five Group 4 (1000 mg/kg) male animals (not statistically significant).

Squamous hyperplasia of the limiting ridge of the forestomach was recorded at minimal or slight degree in four males and in three females of Group 4 (1000 mg/kg) which was statistically significant - p < 0.001 in males and p < 0.01 in females.

There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance.

A moderate degree of fat necrosis was the histological correlate to the nodule seen in the abdominal cavity of one Group 2 (100 mg/kg) rat at necropsy. This finding and the remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all animals assessed.

OTHER FINDINGS

Refer to Sections 7.8.1 and 7.8.2 for details on reproductive and developmental effects.

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Squamous hyperplasia of the limiting ridge of the forestomach at minimal to slight degrees.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: General lack of significant systemic toxicity at the highest dose tested.

Key result

Critical effects observed:

no

Selected clinical biochemistry results:

		Control	100 mg/kg	300 mg/kg	1000 mg/kg
Males (sample of N=5)
Total Protein (g/L)	Mean (+/- sd)	64.7 +/- 2.3	64.9 +/- 1.5	66.5 +/- 1.3	60.5 +/- 1.4*
Albumin (g/L)	Mean (+/- sd)	32.2 +/- 0.7	32.5 +/- 0.8	33.1 +/- 0.5	30.7 +/- 1.1*
Glucose (mmole/L	Mean (+/- sd)	7.99 +/- 0.42	10.81 +/- 2.27*	9.93 +/- 2.01	11.44 +/- 1.35*
Females (sample of N=5)
Total Protein (g/L)	Mean (+/- sd)	66.5 +/- 3.9	64.1 +/- 1.2	65.2 +/- 1.4	62.4 +/- 0.8*
Albumin (g/L)	Mean (+/- sd)	34.9 +/- 1.3	32.9 +/- 1.6*	34.5 +/- 1.1	32.5 +/- 0.4*
Creatine (umol/L)	Mean (+/- sd)	45.2 +/- 1.9	44.7 +/- 1.8	44.7 +/- 2.4	40.8 +/- 1.0**

*/** Dunnett−test based on pooled variance significant at 5% (*) or 1% (**) level

Conclusions:

A repeated dose toxicity study in combination with screening for development and reproduction toxicity was performed according to OECD guideline 422 and GLP principles. Based on the results it was concluded that treatment with 5-(Sodiosulfo)isophthalic Acid by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg body weight/day revealed parental toxicity for local stomach effects at 1000 mg/kg bw/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived:
NOAEL: 300 mg/kg for local stomach effects and at least 1000 mg/kg for systemic toxicity.

Executive summary:

A repeated dose toxicity study in combination with screening for development and reproduction toxicity was performed according to OECD guideline 422 and GLP principles. 5-(Sodiosulfo)isophthalic Acid was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 43-53 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

 

Decreased values for total protein (both sexes), albumin (males), creatinine (females) and increased glucose levels (males) were not accompanied by any corroborative findings, and therefore not regarded as toxicologically significant. Morphological alterations were noted in the epididymides, kidneys and stomach at 1000 mg/kg. Sperm granuloma in the epididymides were recorded in all groups of males with a not statistically significant increase in incidence and severity at 1000 mg/kg. This finding is a not uncommon spontaneous alteration in this strain of rat, although the incidence in this study was above background levels. However, as already two incidences were noted in the control group, the increase at 1000 mg/kg was not considered toxicologically significant and rather to have occurred by chance. In the male kidneys, hyaline droplets in cortical tubules were recorded at a slightly increased incidence and severity at 1000 mg/kg. The hyaline droplets were considered to represent alpha2μglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. This protein is not present in higher mammals, including man, and therefore this finding was not considered relevant to humans and will not be taken into account for determination of the NOAEL. Squamous hyperplasia of the limiting ridge of the forestomach was recorded at minor degrees of severity in both sexes at 1000 mg/kg. This finding was regarded as an indication of a slight irritant property of the test item. No treatment-related toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, haematology, and organ weights).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: US EPA OPPTS 870.3650, July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen-5-sulphoisophthalate

EC Number:

228-845-2

EC Name:

Sodium hydrogen-5-sulphoisophthalate

Cas Number:

6362-79-4

Molecular formula:

C8H6O7S.Na

IUPAC Name:

Sodium 3,5-dicarboxybenzenesulfonate

Constituent 2

Reference substance name:

288-845-2

IUPAC Name:

288-845-2

Constituent 3

Reference substance name:

Sodium-3,5-dicarboxybenzenesulfonate

IUPAC Name:

Sodium-3,5-dicarboxybenzenesulfonate

Constituent 4

Reference substance name:

5-(Sodiosulfo)isophthalic acid

IUPAC Name:

5-(Sodiosulfo)isophthalic acid

Test material form:

other: white powder

Details on test material:

- Name of test material (as cited in study report): 5-(Sodiosulfo)isophthalic Acid
- Physical state: white powder
- Analytical purity: 99.4%
- Lot/batch No.: 6F21174000
- Expiration date of the lot/batch: 2 December 2013
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark
- Other: hygroscopic, store in a well-sealed container

Test animals

Species:

rat

Strain:

other: CrI:WI(Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Refer to Section 7.5.1

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Refer to Section 7.5.1

Details on mating procedure:

Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).

Post-mating Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Detection of mating was not confirmed for animal nos. 43 (Group 1), 58 (Group 2) and 62 (Group 3) which did deliver live offspring. The mating date of these animals were estimated at 21 days prior to the actual delivery dates. These days were designated Day 0 post-coitum.

The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Refer to Section 7.5.1

Duration of treatment / exposure:

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43 - 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Refer to Section 7.5.1

Details on study schedule:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 41, 43 (Group 1), 62 , 70 (Group 3) and 77 (group 4) were not dosed during littering.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

PREPARATION OF DOSING SOLUTIONS:

Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.

Doses were administered by plastic feeding tube.

Dose volume: 5 mL/kg

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

Not determined

Sperm parameters (parental animals):

Parameters examined in selected 5 male parental animals:

testis weight, epididymis weight, prostate weight (after at least 24 h fixation), and weight of seminal vesicles including the coagulating glands

Refer to Section 7.5.1 for all histopathological analyses conducted in this study.

Litter observations:

Each litter was examined for the following if practicle and possible:

Mortality / Viability
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs

At least once daily, detailed clinical observations were made for all animals.

Body weights
Live pups were weighed on Days 1 and 4 of lactation.

Sex
Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

Refer to Section 7.5.1 of this IUCLID file

Postmortem examinations (offspring):

Necropsy

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

Refer to Section 7.5.1 of this IUCLID file

Reproductive indices:

Mating index (%): No. females mated / No. females paired x 100
Fertility index (%): No. pregnant females / No. of females placed x 100
Conception index (%): No. pregnant females / No. females mated x 100
Gestation index (%): No. females bearing live pups / No. pregnant females x 100
Duration ofgestation: No. days between confirmation of mating and the beginning of parturition.

Offspring viability indices:

Percentage live males at first litter check.
Percentage live females at first litter check.
Percentage of postnatal loss.
Viability index (%): No. live pups before necropsy / No. of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Refer to Section 7.5.1 of this IUCLID file

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Refer to Section 7.5.1 of this IUCLID file

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Refer to Section 7.5.1 of this IUCLID file

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer to Section 7.5.1 of this IUCLID file

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Refer to Section 7.5.1 of this IUCLID file for a more complete description of results.

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on a lack of significant reproductive effects measured in the study.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

At 1000 mg/kg bw/day, sex ratio of the pups indicated more female than male pups (62% female pups compared to 47% of the control group); this was statistically significantly different from controls.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Reproduction data summary

	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Females paired	10	10	10	10
Females mated	10	9	10	9
Females non-mated	0	1	0	1
Pregnant females	9	9	10	9
Non-pregnant females	1	0	0	0
Females with living pups on Day 1	9	9	10	9
Mating index (%)	100.0	90.0	100.0	90.0
Fertility index (%)	90.0	90.0	100.0	90.0
Conception index (%)	90.0	100.0	100.0	100.0
Gestation index (%)	100.0	100.0	100.0	100.0

Applicant's summary and conclusion

Conclusions:

A screening study for development and reproduction toxicity was performed according to OECD guideline 422 and GLP principles. No significant reproductive toxicity was noted up to a dose of 1000 mg/kg bw/d. The NOAEL for reproductive toxicity was concluded to be1000 mg/kg bw/day.

Executive summary:

A screening study for development and reproduction toxicity was performed according to OECD guideline 422 and GLP principles. No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites). The NOAEL for reproductive toxicity was concluded to be1000 mg/kg bw/day.