reaction mass of disodium;3-[2-(2-carboxylatoethylamino)-ethylamino]propanoate and sodium 3-[(2-aminoethyl)amino]propanoate

Endpoint summary

Description of key information

Oral: In a study according OECD TG 422 in Wistar rats, the NOAEL for general, systemic toxicity of the test item was 100 mg/kg bw/day based on the findings in the target organ kidney. The NOAEL for reproductive performance and fertility of the F0 parental rats and developmental toxicity in the offspring was 1000 mg/kg bw/day, the highest dose tested.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 November 2016 - January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

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:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Do_0164
- Expiration date of the lot/batch: 30 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (RT), under nitrogen
- Stability under test conditions: guaranteed

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 nulliparous and non-pregnant: yes
- Age at study initiation: males: 14 - 15 weeks old, females: about 13 weeks old
- Weight at pre-mating: males: 387 g, females: 215 g
- Fasting period before study: no
- Housing: during study period, rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
- During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
- Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
Pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical and for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the
Environmental Analytics Water/Steam Monitoring of BASF SE.

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

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:

drinking water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water and intensely mixed with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1 g/100mL, 3 g/100 mL and 10 g/100mL
- Dose volume: 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 1 sample was taken from all concentrations for concentration control analyses.
The concentrations of the test item in the samples were calculated by means of their nitrogen content.
Method for concentration control: After combustion of the vehicle in an argon atmosphere with added oxygen, nitrogen compounds were converted to NO, and oxidized by ozone to excited NO2*. This was detected by photometric measurement of chemiluminescence.
Apparatus: Xplorer TN (TE Instruments)

Method of stability analysis: Capillary electrophoresis (CE) with internal standard quantification
Apparatus: Beckman P/ACE MDQ automated capillary electrophoresis system including capillary oven and UV/Vis-detector

Duration of treatment / exposure:

Females: 65 days
Males: 34 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by request of the sponsor.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and thereafter at weekly intervals
The following parameters listed were assessed: abnormal behavior in “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, 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: once a week
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

FOOD CONSUMPTION: Yes
Generally, food consumption was determined once a week for male and female parental animals and F1 rearing 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.

WATER CONSUMPTION: Yes
- Time schedule for examinations: once a week
Exceptions: Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals). Water consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 3, 7 - 10 and 14 - 17.
Water consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10, 10 - 13.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: 5 surviving parental males per group at termination and first 5 females with litters (in order of delivery) per group at PND 14
- Parameters checked: Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood, Reticulocytes, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
- How many animals: 5 surviving parental males per group at termination and first 5 females with litters (in order of delivery) per group at PND 14
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase
(GGT), sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, tital bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: first 5 male and the first 5 female animals with litter per group
- Battery of functions tested: sensorimotor tests, reflex tests, motor activity measurements

HOME CAGE OBSERVATIONS:
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremors, convulsions, abnormal movements, gait

OPEN FIELD OBSERVATIONS:
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined: behavior on removal from cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypy, gait, activity/arousal level, feces (consistency/color) within 2 minutes, urine (amount/color) within 2 minutes, rearing within 2 minutes

IMMUNOLOGY: No

ESTROUS CYCLE
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 2 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice

MALE/FEMALE REPRODUCTION DATA
Mating, fertility and gestation indices (only females) indices were calculated for F1 litters. The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters. The implantations were counted and the postimplantation loss (in %) was calculated.

LITTER DATA
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups. In general, a check was made for any dead or moribund pups twice daily on workdays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PNDs 1-4, 5-7 and 8-13 were determined. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated. On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. The anogenital index will be calculated. The live pups were examined daily for clinical symptoms. The pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Bulbourethral gland (Cowper’s gland), Epididymides, Glans penis, M. levator ani together with M.bulbocavernosus, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed)
The following weights were determined in 5 animals/sex and test group sacrificed on schedule (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

On PND 4 the surplus pups were sacrificed under isoflurane anesthesia by decapitation. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. After sacrifice, the pups were examined externally, eviscerated and their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 13 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

HISTOPATHOLOGY: Yes
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow(femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital, lacrimal glands, Epididymides (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 gland, 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), Target organs, Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all cohabited female F0 parental animals were stained according to Salewski E (1964)), Vagina

Other examinations:

THYROID HORMONES:
Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).

Statistics:

Blood parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for hypothesis of equal medians
Water consumption, food consumption, body weight, body weight change, gestation days, anogenital distance, anogenital index: Simultaneous comparison of all dose groups with the control group using the DUNNETT test for hypothesis of equal means
Mating indices, fertility indices, females mated, females delivering, gestation index, females with stillborn pups, females with all stillborn pups: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions
Mating days until day 0 p.c., % postimplantation loss, pups stillborn, % perinatal loss, nipple development: Pair-wise comparison of dose group with control group using WILCOXON test with BONFERRONI-HOLM adjustment for hypothesis of equal medians
Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index: Pair-wise comparison of dose group with control using WILCOXON test with BONFERRONI-HOLM adjustment for hypothesis of equal medians % live male day x, %live female day x: Comparison of dose group with control group was performed using WILCOXON test hypothesis of equal medians.
Number of cycles, Cycle Length, Rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test . If resulting p-value was equal or less than 0.05, a pair-wise comparison of dose groups with control group was performed using the WILCOXON test hypothesis of equal medians.
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If resulting p-value was equal or less than 0.05, pairwise comparison of each dose group with control was performed using WILCOXON-test for equal medians

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During pre-mating, mating and post-mating nothing abnormal was seen in all animals of all test groups (100, 300 and 1000 mg/kg bw/d).
At the end of gestation (GD 23) one female animal of test group 3 (1000 mg/kg bw/d) showed dystocia. This animal showed following clinical signs: hypothermia, piloerection, pale skin, blood in bedding in the afternoon of GD 23 and bloody vaginal discharge on GD 24. On PND 0 still bloody vaginal discharge was observed in this female animal. Furthermore all pups of this animal were stillborn and therefore it had complete litter loss. From PND 1 onwards this animal was without any abnormal signs. These signs are considered to be spontaneous in nature and not treatment-related. One female with all pups stillborn is in the range of historical control data.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related differences in mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups were observed in comparison to the concurrent control values during the entire study period. All isolated increases or decreases of body weight and body weight change at isolated time points or intervals of observations showed no dose-dependency and were assessed as spontaneous in nature and not related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of the F0 males and females in all dose groups (100, 300 and 1000 mg/kg bw/day) was not influenced by the treatment throughout the entire study period.
The increased food consumption in the F0 females of test group 2 (300 mg/kg bw/day; 24.4%) and test group 1 (100 mg/kg bw/day; 21.2%) towards the end of lactation (PND 10 to 13) as well as the decreased food consumption in F0 females of test group 3 (1000 mg/kg bw/day) from PND 7 to 10 (-10.5%) were considered as spontaneous in nature and not treatment-related.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

The increased water consumption in the F0 males of test group 3 (1000 mg/kg bw/day; 17.5% and 11.0%) and of test group 2 (300 mg/kg bw/day; 15.6%) during pre-mating and the increased water consumption in females of test group 3 (1000 mg/kg bw/day; 19.5% and 20.6%) during premating and during lactation (PND 4 to 13; maximum 13.1%) were considered as treatment-related but not adverse.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. In males of test group 3 (1000 mg/kg bw/day) absolute large unstained cell (LUC) counts were significantly increased, but the values were within the historical control range (LUC 0.01-0.05 Giga/L). In females of test group 1 (100 mg/kg bw/day) mean corpuscular hemoglobin concentration (MCHC) was higher compared to controls, but the alteration was not dose-dependent. Therefore, both mentioned changes 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 males of test group 3 (1000 mg/kg bw/day) total bilirubin levels were significantly increased, but the values were within the historical control range (total bilirubin 0.90-1.96 μmol/L). In males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/day) inorganic phosphate levels were significantly higher compared to controls. All values were within the historical control range (inorganic phosphate 1.45-1.98 mmol/L). Therefore, both mentioned changes were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. No significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.
Quantitative Parameters:
Following non-significant alterations were assessed as spontaneous in nature and not related to treatment, because of missing dose-dependency: The number of rearings reduced in males of test group 3 (1000 mg/kg bw/day; -44%), test group 2 (300 mg/kg bw/d; -23%) and test group 1 (100 mg/kg bw/day; -38%) and in females of test group 2 (300 mg/kg bw/day; -18%). Increased grip strength of hindlimbs was seen in males of test group 3 (1000 mg/kg bw/day; 14.1%) and females of test group 2 (300 mg/kg bw/day; 12.6%) and test group 1 (100 mg/kg bw/day; 16.1%). In females of test group 1 the grip strength of the forelimbs was also increased (14.7%) A decrease of grip strength of the hindlimbs was observed in males of test group 1 (100 mg/kg bw/day; -20.1%). The foot splay test showed decreased results in females of test group 2 (300 mg/kg bw/day; -11.0%).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean absolute and relative thymic weight changes were noted in males of all groups and females treated with 100 and 1000 mg/kg bw/day. Because of the absence of a dose-relationship, absence of relevant histopathologic changes, and the fact that all mean weights felt within the historical control limits, the thymic weight changes are considered to be of no significant toxicologic relevance.
A slightly increased mean absolute and relative uterus weight was present in females treated with 100, 300 and 1000 mg/kg bw/day. The increase in absolute uterine weight was statistically significant in females of the 300 and 1000 mg/kg bw/day treated group, while the increase in relative weight was only statistically significantly in females of the 1000 mg/kg bw/day treated group.
All other organ weight and/or organ weight ratio changes were not statistically significantly deviating from controls and were attributed to the normal biological variation in animals of this strain and age.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic findings reported are considered spontaneous and consistent with the usual pattern of findings in animals of this strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

During histopathological investigation, in the kidney of a single male and female treated with 300 mg/kg bw/day and in 7 out of 10 males and 5 out of 10 females treated with 1000 mg/kg bw/day test item, papillary necrosis was induced. Next to the incidences, the severity also showed a dose-relationship. In general, renal papillary necrosis is caused by alterations in circulation causing local ischemia resulting in necrosis of vascular and ductal structures and considered an adverse finding. In addition, an increased incidence of minimal to slight tubular basophilia was present in both males and females treated with 1000 mg/kg bw/day. Because of the low severity and predominant unilateral distribution this change is considered to be non-adverse.
Thorough microscopic examination of the male and female reproductive organs did not reveal any relevant histopathologic change. The morphology and distribution of the different successive stages during spermatogenesis was normal for all males examined.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

ESTRUS CYCLE
Estrous cycle data revealed regular cycles in the parental females of all test groups including the control. The mean estrous cycle durations in the different test groups (0-3) were between 3.85 and 3.95 days.
SPERMATOLOGY
The spermatogenic staging profiles were normal for all males examined.
REPRODUCTION DATA
Males: For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups. Fertility was also proven for all F0 parental males of control group and test group 2 (300 mg/kg bw/d) within the scheduled mating interval for F1 litter. Thus, the male fertility index was also 100% for these test groups. The male index for test groups 1 (100 mg/kg bw/day) and 3 (1000 mg/kg bw/day) was 90%, because of only 9 males with pregnant females in these groups within the scheduled mating interval. These values are well within the range of historical control data (70% - 100%)
Females:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.0 / 3.8 / 2.3 /2.7 (test groups 0 – 3). All female rats of test group 0 and 2 (300 mg/kg bw/day) delivered pups. In test group 1 (100 mg/kg bw/day) 8 females and in test group 3 (1000 mg/kg bw/day) 9 females delivered pups. The fertility index was 100% in test groups 0 and 2. In test groups 1 and 3 the female fertility index was 90%. The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.6 days). The gestation index was 100% in test groups 0 and 2. The gestation index in test groups 1 and 3 was 88.9%. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.1 / 12.4 / 13.3 and 14.2 implants/dam in test groups 0 - 3,respectively). The postimplantation loss did not show any significant differences between test groups 0 – 3, and the mean number of F1 pups delivered per dam remained unaffected (11.8 / 12.5 / 12.3 and 11.1 pups/dam in test groups 0 – 3, respectively). For this reason, there were no indications for test substance-induced intrauterine embryo-/fetolethality. The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.2% / 100% / 100% and 98.0% in test groups 0 – 3. The perinatal loss in test
group 3 (1000 mg/kg bw/d) was 11.1%. This relatively high value was caused by one dam having a litter size of two and losing both pups during birth. This finding was within the range of the historical control data (0 – 17.2%, see PART III, Supplement) and were assessed as spontaneous and not treatment-related.
Moreover, the number of stillborn pups was not significantly different between the test groups. Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.
LITTER DATA
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 98.2% / 100% / 97.8% and 86.7% in test groups 0, 1, 2 and did not show any significant difference to the current control. However, the value of test group 3 (1000 mg/kg bw/day) was below the lower end of the istorical control range (89.4 - 100%). The difference was caused by only dam which had only 2 live born pups which both died within postnatal day 1-4 resulting in a pup viability of 0% whereas all other dams with viable pups on PND 0 had a pup viability of 100%. This isolated finding of on dam was assessed as incidental and not related to treatment. The survival index indicating pup mortality on PND 4 – 13 varied between 100% / 100% / 100% and 100% in test groups 0, 1, 2 and 3 without showing any relation to the treatment.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and test group 1. The sex distribution and sex ratios of test groups 2 (300 mg/kg bw/day) and 3 (1000 mg/kg bw/day) showed significant differences towards the control group as the ratio of male pups was increased (57.9% and 60.4%). These values were within the range of the historical control data 36.6-60.5%.
There were no test substance-related, adverse clinical signs observed in any of the other F1 generation pups of the different test groups.
Mean body weights and body weight change of all male and female pups of all test substance-treated groups (100, 300 and 1000 mg/kg bw/day) were comparable to the current control values throughout the entire lactation period. The significantly increased body weights and body weight changes of pups of test group 2 on PND 13 (8.6%*) was an isolated finding without dose-dependency. It was considered as spontaneous in nature and not treatment-related. One female runt each in test group 0 and 2 was seen.
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 male and female pups. The significantly increase of anogenital distance as well as anogenital index cubic root in male pups of test group 2 (300 mg/kg bw/day; 5.4%; 2.75%) was an isolated finding without dosedependency. It was considered to be spontaneous in nature and not related to treatment. Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 male and female pups (test groups 1 – 3 [100 – 1000 mg/kg bw/day]).
The significantly increase of anogenital distance as well as anogenital index cubic root in male pups of test group 2 (300 mg/kg bw/day; 5.4%; 2.75%) was an isolated finding without dose-dependency. It was considered to be spontaneous in nature and not related to treatment.
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13. A few F1 pups showed spontaneous findings at gross necropsy, such as extended intestine and discolored lung. These findings occurred without any relation to dosing and are considered to be spontaneous in nature.
THYROID HORMONES
In parental males of test group 3 (1000 mg/kg bw/day) T4 values were significantly higher compared to controls. However, the mean was within the historical control range (males T4, 44.87-88.29). Therefore, this change was regarded as incidental and not treatment-related. In male and female pups at PND13 (test groups 11, 12 and 13; 100, 300 and 1000 mg/kg bw/day), no treatment-related alterations of T4 levels were observed.

Key result

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance, fertility, developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Stability analysis

The stability of test substance in drinking water was demonstrated for a period of 7 days at room temperature.

Concentration control analysis

The concentrations of the test item in drinking water were found to be in the range of 91 to 101% of the nominal concentration at two independent time points. Analyses demonstrated a recovery of 101% in the low dose (100 mg/kg bw/day), 97% in the mid dose (300 mg/kg bw/day) and 96% in the high dose (1000 mg/kg bw/d) at the beginning of gestation. At the end of gestation a recovery of 101% in the low dose (100 mg/kg bw/day), 95% in the mid dose (300 mg/kg bw/day) and 91% in the high dose (1000 mg/kg bw/d) was demonstrated.

After the analyses of samples collected at the beginning of gestation, the analyses of the samples collected during lactation period were performed and showed a recovery of 71% in the low dose (100 mg/kg bw/d), 99% in the mid dose (300 mg/kg bw/day) and 75% in the high dose (1000 mg/kg bw/day). Since these results did not confirmed the aimed concentration of test substance preparations, the corresponding reserve samples of this test substance preparation were analyzed. In case of comparable analytical results to the one of the main samples, a mistake in the test substance preparation could not be excluded. In case of different analytical results, a mistake analytic procedure would be likely. The reserve samples at the same time point demonstrated a recovery of 100% in the low dose (100 mg/kg bw/day), 57% in the mid dose (300 mg/kg bw/day) and 34% in the high dose (1000 mg/kg bw/day). These results confirmed neither the aimed concentration in the test substance preparation nor the concentration determined in the main samples of the same time point before. The results of the reverse samples were even contradictory. The author assessed the measurements of this time point as biased by problems in analyses. Therefore, the analyses of samples collected during lactation were judged as not suitable to be used for the concentration control. The analyses of further samples of the study were initiated, samples from a second independent test substance preparation during gestation. As described, these results were in good compliance with the expected values.

Food analyses

With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable. The concentration of microorganisms did not exceed 1x10E5/g feed.

Drinking water analyses

On the basis of the analytical findings, the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, 05 Dec 1990) served as a guideline for maximum tolerable contaminant.

Bedding and Enrichment analyses

On the basis of the analytical findings, bedding and cage enrichment were found to be suitable.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD TG 422

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a study according to OECD TG 422 the test item was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg bw/day. Control animals were dosed daily with the vehicle drinking 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 7 days post-mating in males, the entire gestation period as well as up to 13 days of the lactation period in females up to one day prior to the day of schedule sacrifice of the 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.

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) 1, 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 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 stability of the test substance in drinking water was demonstrated over a period of 7 days at room temperature.

In all dose groups no test substance-related adverse findings of P0 parental animals regarding clinical examination, reproductive performance and clinical pathology were reported. In pathology, papillary necrosis in the kidneys of 7 out of 10 males and 5 out of 10 females was detected at the highest dose group of 1000 mg/kg bw/day. At 300 mg/kg bw/day papillary necrosis in the kidneys of one out of 10 males and one out of 10 females was observed. No substance-related adverse findings were detected in the F1 pups at all dose levels.

Under the conditions of the combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL for general, systemic toxicity of the test item was 100 mg/kg bw/day based on the findings in the target organ kidney. the NOAEL for reproductive performance and fertility of the F0 parental rats and developmental toxicity in the offspring was 1000 mg/kg bw/day (BASF SE, 2018).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result, the substance is not considered to be classified for repeated oral dose toxicity, as amended for the tenth time in Regulation (EU) No 2017/776.