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Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Additional information

There are no specific experimental studies concerning fertility available. However, there is sufficient evidence from histopathology data and organ weights of sexual organs (testes, epididymides, seminal vesicles with coagulation glands, vagina, uterus with cervix, ovaries) from a 28-day repeated dose toxicity study with oral (gavage) application in rats (BASF SE, 2010) to conclude that there were no treatment-related adverse effects on these organs. Since the 28-day study showed neither relevantly elevated testis or ovary weights nor relevant histopathological alterations in those organs, there is sufficient evidence that effects on reproduction are not to be expected (BAuA Forschungsbericht Fb 984, 2003).


Short description of key information:
There is sufficient evidence from histopathology data and organ weights of sexual organs (testes, epididymides, seminal vesicles with coagulation glands, vagina, uterus with cervix, ovaries) from a 28-day repeated dose toxicity study with oral (gavage) application in rats (BASF SE, 2010) to conclude that effects on reproduction are not to be expected.

Effects on developmental toxicity

Description of key information
In a prenatal developmental toxicity study according to OECD guideline 414, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 12 mg/kg bw/d. (BASF SE, 2014)
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-09-03 - 2014-04-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 22, 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: 143.6 - 192.3 g
- Housing: individually in Makrolon type M III cages
- Diet: ad libitum, Kliba maintenance diet mouse/rat “GLP”, meal (Provimi, Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum, tap water
- Acclimation period: at least 5 days

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
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the period of established stability. For the test substance preparations, the specific amount of test substance were weighed, topped up with drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer. During administration the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 30, 60, 120 mg/100 mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory at the beginning of administration for verification of the concentrations. The samples were also used to verify the homogeneity of the low and the high concentrations (3 and 12 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.
The dosing samples were analysed by UV/VIS chromatography. Analytical verifications of the stability of the test substance in drinking water over a period of a maximum of 7 days at room temperature were carried out prior to the start of the study.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as GD 0 of pregnancy
Duration of treatment / exposure:
from implantation to one day prior to the expected day of parturition (GD 6 to GD 19)
Frequency of treatment:
once daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
0, 3, 6, 12 mg/kg bw/d
Basis:
nominal in water
No. of animals per sex per dose:
25 females
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels in the present study were based on a range-finding experiment, where pregnant female rats were exposed to 10 and 20 mg/kg bw/d by oral gavage on GD 6-19. A dose of 20 mg/kg bw/d caused immoderate maternal toxicity like clinical findings (such as piloerection, persisting salivation, and semiclosed eyelids), reduced food consumption, increased relative liver and reduced spleen weights, reduced protein (albumines and globulines) levels in plasma, as well as a significantly reduced net body weight gain which was about 50% below the concurrent control. Thus, a top dose of 12 mg/kg bw/d (less than a factor of two below a dose which caused excessive maternal toxicity) was regarded to be appropriate to test the reference substance for its potential effects on rat offspring without causing exaggerated toxicity in their mothers.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: gross necropsy was performed
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
For food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight and litter mean placental weight a simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means was performed.
Female mortality, females pregnant at terminal sacrifice and number of litters with fetal findings were evaluated by pairwise comparison of each dose group with the control group using FISHER'S EXACT test (onesided) for the hypothesis of equal proportions.
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter were evaluated by pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians.
Indices:
The following Indices have been determined:
Conception rate (in %), Preimplantation loss (in %) and Postimplantation loss (in %).
Historical control data:
Historical control data was used to compare findings regarding reproduction data of the dams and external malformations and soft tissue malformations as well as soft tissue variations of the fetuses. Furthermore skeletal malformations as well as variations in the fetuses were compared.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 3, 6 or 12 mg/kg bw/d).

DETAILED CLINICAL OBSERVATIONS
All females of the high-dose group (12 mg/kg bw/d) and most females (14 out of 25) of the mid-dose group (6 mg/kg bw/d) showed transient, blue-discolored salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 10 minutes) and was initially observed on GD 7.
Black discolored feces were recorded in all females of the mid- and high-dose groups (6 and 12 mg/kg bw/d) from GD 10 onwards until terminal sacrifice (GD 20). This feces discoloration mirrors the presence of the test substance (or its metabolites) in the gastrointestinal tract. It is not considered as an adverse toxic effect.
Furthermore, low-dose female No. 35 (3 mg/kg bw/d) had a palpable mass on its throat on GD 13-20 and for high-dose female No. 82 (12 mg/kg bw/d) a cataract on its left eye was recorded on GD 18-20. These findings occurred in single animals and were considered as incidental. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female of the low-dose group (3 mg/kg bw/d) during the entire study period.

BODY WEIGHT
The mean body weights and the average body weight gains of the low-, mid- and high-dose rats (3, 6 or 12 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. This includes the statistically significantly increased body weight change value in the mid-dose group on GD 15-17. The corrected body weight gain of test groups 1-3 (3, 6 and 12 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.

FOOD CONSUMPTION
The mean food consumption of the dams in test groups 1-3 (3, 6 or 12 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period.

POST-MORTEM EXAMINATIONS
UTERUS WEIGHT:
The mean gravid uterus weights of the animals of test group 1-3 (3, 6 and 12 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

NECROPSY FINDINGS:
Blue discolored fore stomachs were recorded in one out of 25 mid-dose females (4%) and 8 out of 25 high-dose females (32%), while dark discolored kidneys were seen in 7 high-dose females (28%). Furthermore, dark discolored contents of the intestine were recorded in one mid-dose female (4%) and 7 high-dose females (28%), dark discolored contents of rectum were seen in one mid-dose female (4%) and 11 high-dose females (44%) and dark discolored contents of caecum were recorded in one mid-dose female (4%) and 10 high-dose females (40%).
These blue or dark discolorations mirror the systemic availability of the test substance or its metabolites. They are not considered as an adverse, toxic effect by themselves.
No necropsy findings which could be attributed to the test substance were seen in any dam of test group 1 (3 mg/kg bw/d). Furthermore, a number of spontaneous findings were noted in individual females of all test groups including the controls (0, 3, 6 or 12 mg/kg bw/d). These gross findings were:

• a palpable mass on the throat in low-dose female No. 35 (left side, diameter: 3 cm, beige-brown color),
• a cataract in high-dose female No. 82 (left eye),
• erosion in stomach in mid-dose female No. 60 and
• dilated renal pelvis in control female No. 8 and high-dose female No. 92 on right side, respectively.

REPRODUCTION DATA:
The conception rate varied between 96% in test groups 1 and 3 (3 and 12 mg/kg bw/d) and 100% in test groups 0 and 2 (0 and 6 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study (according to the used test guidelines). There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 3, 6 and 12 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Dose descriptor:
NOAEL
Effect level:
12 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
12 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
SEX DISTRIBUTION
The sex distribution of the fetuses in test groups 1-3 (3, 6 and 12 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

WEIGHT OF PLACENTAE AND FETUSES
The mean fetal weights of test groups 1, 2 and 3 (3, 6 and 12 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group. The mean placental weights of the low-, mid- and high-dose groups (3, 6 and 12 mg/kg bw/d) were comparable to the corresponding control group.

FETAL EXTERNAL EXAMINATION
Three external malformations were recorded for three fetuses of the control, the low- and mid-dose group (0, 3 or 6 mg/kg bw/d). For the affected fetuses, these external findings were associated with skeletal malformations. The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
No external variations or unclassified observations were recorded.

FETAL SOFT TISSUE EXAMINATIONS
One control fetus had multiple soft tissue malformations, i.e. a malpositioned liver lobe, diaphragmatic hernia, unexpanded lung and misshapen heart. The overall incidences of soft tissue malformations were comparable to those found in the historical control data.

Some soft tissue variations were detected in all test groups including the control (0, 3, 6 or 12 mg/kg bw/d), i.e. short innominate, malpositioned carotid origin, supernumerary liver lobe and dilated renal pelvis. However, the incidence of dilated renal pelvis was statistically significantly increased in the low-dose group (3 mg/kg bw/d), but it was well within the historical control range (HCD: 2.3% [0.0 – 6.3%]). Thus, all variations were either not statistically significantly different from control or not dose dependent and therefore, not considered biologically relevant.

No unclassified soft tissue observations were recorded.

FETAL SKELETAL EXAMINATION
A number of skeletal malformations were detected in all test groups (0, 3, 6 and 12 mg/kg bw/d) affecting the skull, vertebral column, sternum and forelimbs. One fetus of test groups 0, 1 and 2, respectively, had associated external findings. All other findings were single cases, most of them can be found in the historical control data. An association of these malformations to the treatment is not assumed.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the sternum and ribs and, in most cases, did not show any relation to dosing. However, the incidence of notched cartilage between basisphenoid and basioccipital was statistically significantly increased in test group 3 (12 mg/kg bw/d), and the incidence of cartilaginous parts of ribs displaced was statistically significantly increased in test group 2 (6 mg/kg bw/d). The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.

OVERALL ASSESSMENT
There were noted external, soft tissue and skeletal malformations in all test groups. The distribution of total malformations about the groups was not related to dose. Some fetuses were multiple-malformed. Male fetus No. 18-06 (control group) had several visceral malformations, i.e. a malpositioned liver lobe, a diaphragmatic hernia, an unexpanded lung and a misshapen heart. Furthermore, male fetus No. 47-06 (test group 1 – 3 mg/kg bw/d) had multiple external malformations, such as cleft palate, upper jaw short and pointed, mandible pointed and unilateral anophthalmia, associated with multiple skeletal malformations concerning the whole fetal body. For control female fetus No. 19-11 shortened scapula, shortened humerus and bent radius were recorded. Male fetus No. 18-05 (control group) had a short tail (comprising a severely malformed vertebral column), while female fetus No. 51-10 (test group 2 – 6 mg/kg bw/d) had mandibular micrognathia (comprising a fused and additionally shortened mandible). Other malformations, such as an additional vertebral arch, misshapen tuberositas deltoidea, shortened humerus and malpositioned and bipartite sternebra observed in test groups 0-3 are common for this rat strain and some of them can be found in the historical control data at a comparable or higher frequency. An association of these findings to the treatment is not assumed.
External variations did not occur in any of the fetuses in this study. Four soft tissue variations and a broad range of skeletal variations occurred in all test groups including the controls. None of the incidences showed a clear relation to dosing. The skeletal variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a similar or comparable frequencies.
No unclassified external or soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3 (0, 3, 6 and 12 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. However, the incidence of notched cartilage between basisphenoid and basioccipital was statistically significantly increased in test group 3 (12 mg/kg bw/d). The incidence of this finding slightly exceeds the historical control range (11.4% vs. 10.0% affected fetuses/ litter). However, it is still a very common spontaneous finding in this rat strain and the level of concern for this sort of findings is rather low. Therefore this minor potential effect is assessed to be without biological relevance. Finally, fetal examinations revealed that there is no biological meaningful effect of the compound on any morphological structures up to the highest dose tested (12 mg/kg bw/d).
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
12 mg/kg bw/day
Species:
rat
Quality of whole database:
GLP and guideline compliant study.
Additional information

The reference substance was tested for its prenatal developmental toxicity according to OECD guideline 414 and EU method B.31 (BASF SE, 2014). The test substance was administered as an aqueous suspension to groups of 25 time-mated female Wistar rats by gavage at doses of 3, 6, and 12 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 24-25 females per group had implantation sites. Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, all females were sacrificed by cervical dislocation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and the placentae). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 3, 6 or 12 mg/kg bw/d of the test substance and controls. All females of the high-dose group (12 mg/kg bw/d) and most females (14 out of 25) of the mid-dose group (6 mg/kg bw/d) showed transient, blue-discolored salivation after treatment. This salivation persisted in the respective females for a few minutes immediately after each administration. It is considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. Blue or dark discolorations were found in a number of organs/tissues at necropsy. These discolorations mirror the systemic availability of the test substance or its metabolites. They are not considered as an adverse, toxic effect by themselves. No differences of toxicological relevance between the control and the treated groups (3, 6 or 12 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.

There were noted external, soft tissue and skeletal malformations in all test groups. The distribution of total malformations about the groups was not related to dose. Some fetuses were multiple-malformed. Male fetus No. 18-06 (control group) had several visceral malformations, i.e. a malpositioned liver lobe, a diaphragmatic hernia, an unexpanded lung and a misshapen heart. Furthermore, male fetus No. 47-06 (test group 1 – 3 mg/kg bw/d) had multiple external malformations, such as cleft palate, upper jaw short and pointed, mandible pointed and unilateral anophthalmia, associated with multiple skeletal malformations concerning the whole fetal body. For control female fetus No. 19-11 shortened scapula, shortened humerus and bent radius were recorded. Male fetus No. 18-05 (control group) had a short tail (comprising a severely malformed vertebral column), while female fetus No. 51-10 (test group 2 – 6 mg/kg bw/d) had mandibular micrognathia (comprising a fused and additionally shortened mandible). Other malformations, such as an additional vertebral arch, misshapen tuberositas deltoidea, shortened humerus and malpositioned and bipartite sternebra observed in test groups 0-3 are common for this rat strain and some of them can be found in the historical control data at a comparable or higher frequency. An association of these findings to the treatment is not assumed. External variations did not occur in any of the fetuses in this study. Four soft tissue variations and a broad range of skeletal variations occurred in all test groups including the controls. None of the incidences showed a clear relation to dosing. The skeletal variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a similar or comparable frequencies. No unclassified external or soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3 (0, 3, 6 and 12 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. However, the incidence of notched cartilage between basisphenoid and basioccipital was statistically significantly increased in test group 3 (12 mg/kg bw/d). The incidence of this finding slightly exceeds the historical control range (11.4% vs. 10.0% affected fetuses/ litter). However, it is still a very common spontaneous finding in this rat strain and the level of concern for this sort of findings is rather low. Therefore this minor potential effect is assessed to be without biological relevance.

Finally, under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 12 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.

In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 12 mg/kg bw/d.

Justification for classification or non-classification

Effects on fertility: Based on the available data, there is no concern for toxicity to reproduction. Classification according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008 is therefore not warranted.

Developmental toxicity/teratogenicity: Based on the available data, there is no concern for developmental toxicity. Classification according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008 is therefore not warranted.