Reaction mass of 5,5'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[3-methylsalicylic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and potassium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxy-7-sulfonaphthalene-2-sulfonate - 2,2',2''-nitrilotriethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 7070141023
- Expiration date of the Batch: Jul 2021
- Content: 81.3 g/100 g (100 g/100 g - Neopentyl glycol (5.9 g/100 g) - water (12.8 g/100 g))
- Physical state / appearance: solid / black

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient; avoid temperatures < 0 °C
- Stability under test conditions: The stability of the test substance in deinoized water was demonstrated for a period of 7 days at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparations in deionized water were prepared in intervals, which took into account the analytical results of the stability verification. The test substance preparations were produced weekly, at least. For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with deionized water and mixed with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : suspension

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Details on species / strain selection:

The test guidelines require the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males 70 - 76 days, Females about 77 - 90 days
- Weight at study initiation: The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex.
- Housing: During pretreatment, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages. During the study period, the rats were housed individually in Polycarbonate cages with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages; Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse / rat "GLP" meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): drinking water was supplied from water bottles; ad libitum
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.

Vehicle:

water

Remarks:

deionized

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations in deionized water were prepared in intervals, which took into account the analytical results of the stability verification. The test substance preparations were produced weekly, at least.
For the preparation of the administration suspensions the test substance was weighed in a calbrated beaker depending on the dose group, topped up with deionized water and mixed with a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in deionized water for a period of 7 days atroom temperature were carried out prior to the start of the administration period. At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above-mentioned time points, additionally, one sample from the mid concentration was taken for concentration control analysis. Of each sample, one additional reserve sample (described by the suffix "R") was retained. The samples collected at the beginning and during lactation period were analyzed in the Analytical Laboratory. Additionally, the reserve samples of the highest concentration collected at the end of the study (lactation period) and samples of the highest concentration collected at the end of gestation period were analyzed. All further samples were not analyzed and stored frozen (at - 20 °C) in the Laboratory of the Mechanistic Toxicology until the finalization of the study.

Duration of treatment / exposure:

The duration of treatment covered a 2 weeks premating period and mating period in both sexes (mating pairs were from the same dose group), approximately 3 days post-mating in males, the entire gestation period, up to 13 days of the lactation period in females as well as up to one day prior to the day of schedule sacrifice of animals.

Frequency of treatment:

once daily at approximately the same time in the morning

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

corresponds to 105 mg/kg bw/d main ingredient (content of the main test compound is 87.2 % including 5.9% neopentyl glycol)

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

corresponds to 305 mg/kg bw/d main ingredient (content of the main test compound is 87.2 % including 5.9% neopentyl glycol)

Doses / concentrations 3

Dose / conc.:

1 150 mg/kg bw/day (nominal)

Remarks:

corresponds to 1003 mg/kg bw/d main ingredient (content of the main test compound is 87.2 % including 5.9% neopentyl glycol)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected by request of the sponsor. The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.
- Rationale for animal assignment (if not random): The animals were distributed according to weight among the individual test groups, separated by sex.
- Fasting period before blood sampling for clinical biochemistry: yes, feed was withdrawn for about 16 - 20 hours

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena.
- The following parameters listed were assessed: Abnormal behavior in "handling", fur, skin, posture, salivation, respiration, activity / arousal level, tremors, convulsions, abnormal movements, gait a bnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance / consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female parental animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13. Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION: Yes
- Time schedule: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions: Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20. Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10, 10 - 13. Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week for the male and female parental animals over a period of 3 days. Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals). Water consumption of the females with evidence of sperm was determined for GD 0 - 3, 7 - 10 and 14 - 17. Water consumption of the females which gave birth to a litter was determined for PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13. Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning (males: study day 30, females: PND 14) blood was taken from the retro-bulbar venous plexus from fasted animals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- The following parameters were examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RETA), prothrombin time (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning (males: study day 30, females: PND 14) blood was taken from the retro-bulbar venous plexus from fasted animals.
- Animals fasted: Yes
- The following parameters were examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), sodium (NA), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (CA), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), bile acids (TBA)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period starting at about 10.00 h
- Dose groups that were examined: all dose groups
- Battery of functions tested: home cage observations, open field observations, sensory motor tests / reflexes, motor activity measurement

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy consisted of gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY: Yes
- The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Epididymides
3. Ovaries
4. Prostate (ventral and dorsolateral part together, fixed)
5. Seminal vesicles with coagulating glands (fixed)
6. Testes
7. Thyroid glands (with parathyroid glands) (fixed)
8. Uterus with cervix
- The following weights were determined in 5 animals per sex / test group sacrificed on schedule:
1. Adrenal glands (fixed)
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus (fixed)
- The following organs or tissues of all parental animals were fixed in 4 % neutral buffered formaldehyde solution or in modified Davidson's solution: all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, esophagus, epididymides (modified Davidson's solution), extraorbital lacrimal glands, eyes with optic nerve (modified Davidson's solution), femur with knee joint, heart, ileum, jejunum (with Peyer's Patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson's solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes (modified Davidson's solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
- The eyes with optic nerve and ovaries of the animals that sacrificed intercurrently were fixed in 4 % neutral buffered formaldehyde solution

Statistics:

- DUNNETT-test (two-sided): food consumption (F0), water consumption (F0), body weight and body weight change (F0, F1), gestation days, anogenital distance, anogenital index
- FISHER'S EXACT test (one-sided): male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups
- WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment: mating days until day 0 pc, % postimplantation loss, pups stillborn, % perinatal loss, nipple development
- WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment: implantation sites, pups delivered, pups liveborn, live pups day x, viability index, survival index
- WILCOXON test (two-sided): % live male day x, % live female day x
- KRUSKAL-WALLIS test (two-sided): number of cycles and cycle length, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, weight parameters

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the entire study period (from premating day 2 onwards) black discolored feces were observed in all male and female animals of test group 2 and 3 (350 and 1150 mg/kg bw/d). Furthermore all animals of test group 3 showed grey discolored skin at the entire body from premating day 7 onwards. These findings were considered to be related to treatment, caused by the color of the test compound, and therefore assessed as being not adverse. One female animal of test group 2 (No. 130, 350 mg/kg bw/d) showed semiclosed eyelids, pale skin, piloerection, unsteady gait and hypothermia on gestation day 21, the day before delivery. Since these findings were gone on the next day after delivery it seems obvious that these were caused by problems with littering and therefore not treatmentrelated.

Detailed clinical observations
In all males and females of test group 3 (1150 mg/kg bw/d) discolored skin and discolored feces were observed from day 7 onwards. In test group 2 (350 mg/kg bw/d) all animals showed discolored feces from day 7 onwards. These findings were considered to be treatment-related but not adverse. One female animal (No. 130) of test group 2 additionally showed semiclosed eyelids, pale skin, piloerection, unsteady gait and hypothermia on study day 35 which was the day before delivery. These isolated findings were considered to be caused by problems with littering since they were gone after littering. All male and female animals in test group 1 (120 mg/kg bw/d) did not show any alterations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal (No. 133) of test group 3 (1150 mg/kg bw/d) was sacrificed moribund on gestation day 6 due to clinical findings such as hypothermia, poor general condition, unsteady gate, slightly labored respiration, piloerection, vaginal discharge and heavy discoloration of skin and eyes (black). Considering the heavy discoloration of the animal and the pathology observations the findings were assessed to be caused by a gavage error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights of all male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study period. Body weight changes are staggering throughout all dose groups, partly increased and partly decrease
d. Since there is no dose-effect relationship showing up this was considered to be not treatment related and non adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the F0 animals in all test groups was not influenced by the treatment throughout the entire study period.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

In male animals of test group 3 (1150 mg/kg bw/d) water consumption was significantly increased from premating day 0 to 3 (21.4 %) and a slight (non-statistically significant) increase was still observed from premating day 7 to 10 (16.2 %). Males of test group 2 (350 mg/kg bw/d) showed slightly (non-statistically significant) increased water consumption from premating day 0 to 3 (16.4 %) as well as males of test group 1 (120 mg/kg bw/d) on premating days 0 to 3 (13.0 %) and 7 to 10 (18.4 %). In females of test group 2 a slight (non-statistically significant) increase of water consumption was observed from premating day 7 to 10 (13.4 %). During gestation slightly (non-statistically significant) increased water consumption was observed in test group 3 on days 7 to 10 (13.5 %) and 14 to 17 (12.1 %) whereas animals of test group 1 showed reduced water consumption from day 14 to 17 (- 12 %). In animals of test group 2 increased (non-statistically significant) water consumption was observed on lactation days 4 to 7, 7 to 10 and 10 to 13 (11.5 %, 12 % and 26.5 %) as well as in animals of test group 3 on lactation days 7 to 10 (12.1 %).
The increase of water consumption after the start of administration in the premating phase showed a dose-dependency in the values between day 0 and 3. Afterwards, no clear dose-dependency was given at later time points. Therefore, for the alterations at the beginning of administration it cannot be excluded that the alterations were treatment-related. However, because of temporary occurrence of the alterations in dose-dependency, they were assessed as non-adverse. Due to the sporadic occurrence without dose-dependency, the alterations in water consumptions at later time points during the administration period were considered to be spontaneous in nature and independent of treatment.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed.
At the end of the administration period, in males of test groups 2 and 3 absolute neutrophil cell counts were significantly decreased. Total white blood cell (WBC) counts and absolute lymphocyte counts were also lower in these individuals, although not statistically significantly. WBC and absolute lymphocyte counts in both mentioned test groups and absolute neutrophil cell counts in test group 2 were within historical control ranges, whereas the absolute neutrophil count mean in males of test group 3 was marginally below this range (males, total WBC 4.50 - 7.04 Giga/L; absolute lymphocytes 3.38 - 5.96 Giga/L; absolute neutrophils 0.73 - 1.44 Giga/L). In females, no changes of the differential blood cell counts were observed. Therefore, the mentioned changes in males of test groups 2 and 3 were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.
In dams at lactational day 14, sodium levels were significantly increased, but the mean was only marginally above the historical control range (females, sodium 132.9 - 139.8 mmol/L) and this was the only changed electrolyte value. Therefore, this alteration was regarded as incidental and not treatmentrelated.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observation: In the open field observations each one male animal of test group 2 (350 mg/kg bw/d; No. 23) and test group 3 (1150 mg/kg bw/d; No. 32) showed grey discolored urine. This finding was considered to be treatment-related but not adverse. Concerning all other animals the open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests / reflexes: There were no test substance-related findings in male and female animals of all test groups.
- Quantitative parameters: There were no test substance-related findings in male and female animals of all test groups.
- Motor activity measurement (MA): No treatment-related changes on motor activity data (summation of all intervalls) were observed in all male and female animals of all dose groups in comparison to the concurrent control group. In female animals of test group 3 (1150 mg/kg bw/d) a significantly increase of beam interruptions was recorded for interval 4, 5 and 8. Furthermore in test group 2 (350 mg/kg bw/d) a significantly decrease of beam interruptions was recorded for interval 2 as well as in test group 1 (120 mg/kg bw/d) for interval 12. These isolated single findings in test group 1 - 3 without any effect on the respective sum of all intervals, were assessed as incidental and spontaneous in nature.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute organ weights
When compared to control group 0 (= 100 %), the mean absolute weights of kidneys in male animals of test groups 2 and 3 were significantly increased. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (= 100 %), the mean relative weights of kidneys in male animals of test groups 2 and 3 and in female animals of test group 3 were significantly increased. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The significant increases of the kidneys weights in test group 3 males (absolute and relative) and female (relative) were consistent with histopathological findings and were regarded as treatment-related. The significant absolute and relative weight increases in test group 2 males (absolute: 2.73 g; relative: 0.73 %) were both minimally above the historical control range (absolute: 2.264 - 2.674 g; relative: 0.564 - 0.652 %). Although no histopathological correlate was found, the weight increase was regarded as treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Grey discolorations were seen in several organs of all males and females of test group 3 (forestomach, glandular stomach, jejunum, cecum, colon, liver, pancreas, mesenteric lymph node, epididymides, prostate, testes, ovaries, uterus, skin) and single animals of test group 2. In test group 1, only the kidneys showed grey discolorations in males and females. The discolorations were considered treatment-related and most likely reflected the capacity of the test substance to stain tissues. However, no histopathological correlate was seen in most of the organs, since the grey discolorations were most likely washed out during the histotechnical processing of the tissues.
The animal sacrificed in a moribund state (No. 133) showed a black discoloration in all tissues, organs and gastrointestinal contents. A black effusion was also found in the thoracic cavity.
All other findings occurred individually and were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animal No. 129 which was not pregnant as well as the male mating partner No. 29 did not show relevant gross lesions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the kidneys of males and females.
In test group 3 males and females, a minimal to moderate degeneration / regeneration of proximal convoluted tubules was noted and was associated with an intracellular basophilic granular pigmentation (minimal to slight). In test group 2 males and females, only a minimal accumulation of basophilic pigmentation occurred. No pigmentation was detected in test group 1. A special stain proved that the nature of these granular basophilic pigmentation was not a dystrophic mineralization (von Kossa stain negative). The pigmentation seen in the kidneys by light microscopy might be directly correlated to the test substance but it cannot be further characterized chemically by histopathological methods.
The female of test group 3, sacrificed in a moribund state (No. 133) showed a severe tubular necrosis accompanied by a severe basophilic pigmentation not only in cortical tubular cells but also in the lumina of cortical, medullar and papillary tubules. In addition, basophilic emboli were found in glomerular capillaries and interstitial vessels. In the lungs, basophilic emboli of the same nature as described in the kidneys were detected in the venules. In the heart, a slight hemorrhage was noted at the base of the heart, around the great vessels and partly in the myocardium, accompanied here by a minimal focal necrosis. The lesions in the base of the heart and the black effusion in the thoracic cavity found at gross pathology are assumed to be the result of a gavage error.
All other findings occurred either inidvidually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
The female animal No. 129, which was not pregnant as well as the male mating partner No. 29 did not show relevant histopathological findings that could have impaired the fertility.

Histopathological findings: neoplastic:

no effects observed

Details on results:

- The male mating index was 100 % in test groups 0, 1 and 3. In test group 2 the male mating index was 90 % due to male No. 29 mated with female No. 129 without evidence of sperm within the mating period.
- Fertility was proven for nearly all of the F0 parental males within the scheduled mating interval to produce F1 litter with exception of one male in test group 2. This male of test group 2 (No. 129 mated with female No. 129) did not generate implants in utero. The male fertility index was 100 % in test groups 0, 1 and 3 and 90 % in test group 2. Thus, the test substance did not affect fertility of the F0 generation parental males.
- The female mating index calculated after the mating period for F1 litter was 100 % in test groups 0, 1 and 3 and 90 % in test group 2. One female (No. 129) of test group 2 showed no evidence of sperm within the scheduled mating period. The mean duration until sperm was detected (GD 0) was 2.4, 2.2, 4.8, 2.7 days in test groups 0 - 3. This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- All sperm positive rats got pregnant. The female fertility index was 100 % in all test groups. The mean duration of gestation was similar in all test groups (22.1 days in test group 0; 21.9 days in test group 1; 22.0 days in test group 2; 22.2 days in test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- The gestation index was 90 % in the control group and 100 % in all treated groups.
- The rate of live birth indices were 99.1 % in test group 0, 98.3 % in test group 1, 100 % in test group 2 and 97.1 % in test group 3. There was 1 stillborn pup recorded in control group (0.9 %), 2 stillborn pups recorded in test group 1 (1.7 %) and 3 stillborn pups recorded in test group 3 (2.9 %). The values of test groups 1, 2 and 3 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (pups liveborn 91.7 - 100 %, pups stillborn 0.0 - 8.3 %). Thus, the test substance did not affect fertility, reproduction and delivery of the F0 generation parental females.
- The postimplantation loss was 11.5 % in test group 0, 0 % in test group 1, 6.2 % in test group 2 and 10.6 % in test group 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (0.0 - 18.1 %).

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: general systemic toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

305 other: mg/kg bw/day (active ingredient)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: general systemic toxicity

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

1 150 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 003 other: mg/kg bw/day (active ingredient)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1: Male fertility indices for F0 males

	Test group 0 (0 mg/kg bw/d)	Test group 1 (120 mg/kg bw/d)	Test group 2 (350 mg/kg bw/d)	Test group 3 (1150 mg/kg bw/d)
Male fertility index [%]	100	100	90	100

* p≤0.05; ** p≤0.01

Table 2: Sex ratio of live F1 pups

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (120 mg/kg bw/d)	Test group 2 (350 mg/kg bw/d)	Test group 3 (1150 mg/kg bw/d)
Live males [%]	48.8	56.6	51.4	54.7
Live females [%]	51.2	43.4	48.6	45.3
PND 13
Live males [%]	50.8	51.7	50.0	50.8
Live females [%]	49.2	48.3	50.0	49.2

* p ≤ 0.05; ** p ≤ 0.01

Table 3: Absolute organ weights of F0 parental animals

	Male animals
Test group (mg/kg bw/d)	1 (120)	2 (350)	3 (1150)
Kidneys	+2.83%	+13.58%*	+20.58%**

*p <= 0.05; **p <= 0.01

Table 4: Relative organ weights of F0 parental animals

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (120)	2 (350)	3 (1150)	1 (120)	2 (350)	3 (1150)
Kidneys	+4.46%	+15.14%*	+21.49%**	+2.98%	+8.20%	+18.06%**

*p <= 0.05; **p <= 0.01

Table 5: Histopathology of F0 parental animals

	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (120)	2 (350)	3 (1150)	0 (0)	1 (120)	2 (350)	3 (1150)
No. of animals	10	10	10	10	10	10	10	10
Degeneration / regeneration, tubules, mf	0	0	0	10	0	0	0	9
-      Grade 1				1				1
-      Grade 2				5				4
-      Grade 3				4				4
Pigmentation, tubular, mf	0	0	3	10	0	0	7	10
-      Grade 1			3	4			7	1
-      Grade 2				6				8
-      Grade 4								1

Applicant's summary and conclusion

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed treatment-related adverse systemic findings in parental animals (F0) manifested in the kidneys of both sexes at 1150 mg/kg bw/day.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 350 mg/kg bw/d in both sexes of parental animals. The no observed adverse effect level (NOAEL) for reproductive performance and fertility was 1150 mg/kg bw/day in both sexes of parental animals.
The NOAEL for developmental toxicity in the F1 progeny was 1150 mg/kg bw/day.

Executive summary:

The test substance was administered daily as a liquid preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at nominal doses of 120, 350 and 1150 mg/kg bw/d. Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (deionized water). The duration of treatment covered a 2 weeks premating period and mating period in both sexes (mating pairs were from the same dose group), approximately 3 days post-mating in males, the entire gestation period, up to 13 days of the lactation period in females as well as up to one day prior to the day of schedule sacrifice of all animals.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Water consumption of the F0 parents was determined once weekly over a period of 3 days during premating. In dams, water consumption was determined for GD 0 - 3, 7 - 10, 14 - 17 and PND 1 - 4, 4 - 7, 7 - 10, 10 - 13. Food consumption of the F0 parents was determined once weekly during premating. In dams, food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10, 10 - 13. Body weights of F0 parents were determined once a week. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20 and on postnatal days (PND) 0, 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. At necropsy on PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation, all other pups (on PND 13) were sacrificed with CO2, under isoflurane anesthesia. All pups were examined macroscopically for external and visceral findings. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving male pups were examined for the presence or absence of nipple / areola anlagen on PND 13. The number of nipple / areola anlagen were counted. Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for determination of thyroid hormone concentrations. Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. At the end of the administration period a functional observational battery was performed, and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded, and a histopathological examination was performed.

The various analyses confirmed the stability of the test substance in deionized water over a period of 7 days at room temperature and confirmed the overall accuracy of the prepared concentrations.

The following test substance-related adverse effects / findings were noted:

Test group 3: 1150 mg/kg bw/d (corresponding to 1003 mg/kg bw/d active ingredient)

F0 Parental Animals

Clinical examinations, reproductive performance, and clinical pathology

- No test substance-related adverse findings

Pathology

- Statistical significant kidney weight increase in males: absolute (+ 20.58 %) and relative (+ 21.49 %), and females: relative (+ 18.06 %)

- Degeneration-regeneration of tubules in kidneys: all males and 9 out of 10 females (minimal to moderate)

- Tubular rigmentation in kidneys of all males and females (minimal to slight)

F1 Pups

Clinical examinations / gross findings and clinical pathology

- No test substance-related adverse findings

Test group 2: 350 mg/kg bw/d (corresponding to 305 mg/kg bw/d active ingredient)

F0 Parental Animals

Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 Pups

Clinical examinations / gross findings and clinical pathology

- No test substance-related adverse findings

Test group 1: 120 mg/kg bw/d (corresponding to 105 mg/kg bw/d active ingredient)

F0 Parental Animals

Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 Pups

Clinical examinations / gross findings and clinical pathology

- No test substance-related adverse findings

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed treatment-related adverse systemic findings in parental animals (F0) manifested in the kidneys of both sexes at 1150 mg/kg bw/day (i.e. 1003 mg/kg bw/day active ingredient).

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 350 mg/kg bw/d (i.e. 305 mg/kg bw/d active ingredient) in both sexes of parental animals. The no observed adverse effect level (NOAEL) for reproductive performance and fertility was 1150 mg/kg bw/day (i.e. 1003 mg/kg bw/day active ingredient) in both sexes of parental animals.

The NOAEL for developmental toxicity in the F1 progeny was 1150 mg/kg bw/day (i.e. 1003 mg/kg bw/day active ingredient).