Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

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.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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
Qualifier:
according to
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. certificate)
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 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
Vehicle:
water
Remarks:
drinking water
Details on 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
- Any other deviations from standard protocol: no
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
Parental animals: Observations and examinations:
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:
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.
Oestrous cyclicity (parental animals):
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
Sperm parameters (parental animals):
Parameters examined in P0 male parental generation:
testis weight, epididymis weight, prostate weight, seminal vesicles with coagulating glands
Special attention was given to the stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups, organs were assessed macroscopically, thyroid hormones (T4)

GROSS EXAMINATION OF DEAD PUPS: yes
for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
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

The following weights will be determined in all animals sacrificed on schedule: anesthetized animals, epididymides, ovaries, prostate, seminal vesicles with coagulating glands, testes, thyroid glands (fixed), uterus (with cervix)
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands, brain, heart, kidneys, liver, spleen, thymus
Postmortem examinations (offspring):
SACRIFICE
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation.

GROSS NECROPSY
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
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Thyroid glands/parathyroid glands were fixed in neutral buffered 4%formaldehyde solution and analysed.
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 the 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 footsplay 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
Reproductive indices:
For the males, mating and fertility indices were calculated for F1 litters.
For the females, mating, fertility and gestation 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.
Offspring viability indices:
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 (lactation period) were determined. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7 and 13. Furthermore, viability and lactation indices were calculated. The sex ratio was calculated at PND 0 and PND 13.
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 inbedding 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/day; -23%) and test group 1 (100 mg/k
g 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
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 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):
In parental males of test group 3 (1000 mg/kg bw/d) 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.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
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.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The spermatogenic staging profiles were normal for all males examined.
Reproductive performance:
no effects observed
Description (incidence and severity):
Fertility was also proven for all F0 parental males of control group and test group 2 (300 mg/kg bw/day) 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%).
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.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
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
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related, adverse clinical signs observed in any of the other F1 generation pups of the different test groups
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
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 historical 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.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
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%. Therefore, these findings were assessed as incidental 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.
In male and female pups at PND13 (test groups 100, 300 and 1000 mg/kg bw/day), no treatment-related alterations of T4 levels were observed.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance, fertility, developmental toxicity
Generation:
F1
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:
no
Key result
Reproductive effects observed:
no

Table 1: Summary Pregnancy Status Report - Reproduction

 

 

Test Group 0
0 mg/Kg bw/d

Test Group 1
100 mg/kg bw/d

Test Group 2
300 mg/Kg bw/d

Test Group 3
1000 mg/Kg bw/d

No. of females at start

N

10

10

10

10

No. of females mated

N

10

10

10

10

Without evidence of mating

N

0

0

0

0

- Pregnant

N

0

0

0

0

- Not pregnant

N

0

0

0

0

Females with defined Day 0 pc

N

10

10

10

10

Pregnant

N

10

9

10

9

- sacrificed scheduled

N

10

9

10

9

Not pregnant

N

0

1

0

1

- sacrificed scheduled

N

0

1

0

1

Pregnant, not delivering

N

0

1

0

0

Delivehng

N

10

8

10

9

- With liveborn pups

N

10

8

10

8

 

%

100.0

100.0

100.0

88.9

- With all pups stillborn

N

0

0

0

1

 

%

0.0

0.0

0.0

11.1

 

Table 2: Summary Mating Report

 

 

Test Group 0
0 mg/Kg bw/d

Test Group 1
100 mg/Kg bw/d

Test Group 2
300 mg/Kg bw/d

Test Group 3
1000 mg/Kg bw/d

No. of females mated

N

10

10

10

10

- Inseminated

N

10 f-

10

10

10

Female mating index

%

100.0

100.0

100.0

100.0

- Pregnant

N

10 f-

9

10

9

Female fertility index

%

100.0

90.0

100.0

90.0

No. of males mated

N

10

10

10

10

- With inseminated females

N

10 f-

10

10

10

Male mating index

%

100.0

100.0

100.0

100.0

-With pregnant females

N

10 f-

9

10

9

Male fertility index

%

100.0

90.0

100.0

90.0

Females with defined Day 0 pc

N

10

10

10

10

Mating days until Day 0 pc

Mean

2.0 x+

3.8

2.3

2.7

 

S.d.

0.9

3.6

1.1

1.1

Days 0 To 4

N

10

9

10

10

 

%

100.0

90.0

100.0

100.0

Days 5 To 9

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

Days 10 To 14

N

0

1

0

0

 

%

0.0

10.0

0.0

0.0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided-*-), * p<=0.05, ** p <=0.01

X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

 

Table 3: Histopathology incidence table: Treatment-related findings

 

DOSE GROUP

0

 

100

 

300

 

1000

 

 

NO. ANIMALS

10 (M)

10 (F)

10 (M)

10 (F)

10 (M)

10 (F)

10 (M)

10 (M)

  

KIDNEYS

10

10

10

10

10

10

10

10

 

N.A.D

 2

 7

 6

 8

 6

 6

 1

 2

-- Tubular basophilia

  

 

-

2

-

1

1

2

6

6

 

Grade 1

   

-

2

-

1

1

1

4

5

Grade 2

   

-

-

-

-

-

1

2

1

-- Papillary necrosis

   

-

-

-

-

1

1

7

5

 

Grade 1

   

-

-

-

-

1

1

1

1

 

Grade 2

   

-

-

-

-

-

-

6

4

 

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/day) 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/day) 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/day), 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD TG 422
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a study according 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. 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.

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).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD TG 422
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

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. There is no evidence of reproductive or developmental toxicity. As a result, the substance is not considered to be classified for developmental toxicity, as amended for the tenth time in Regulation (EU) No 2017/776.