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EC number: 240-457-5 | CAS number: 16409-43-1
Based on the results of this range-finding study, dose levels of 50, 250 and 800 mg/kg bw/d have been selected for the main developmental toxicity study acc. to OECD 414 (BASF 2019; 30R0624/07R071).
In the maternal toxicity dose range-finding study dose levels of 500 and 1000 mg/kg bw/d were tested (BASF 2019; 10R0624/07R067). The test substance was administered to pregnant female Wistar rats on GD 6 through GD 19. In this study clinical signs like transient salivation and animals ploughing their nose into bedding indicated a bad taste and/or a slightly irritating potential of the test item in the upper gastrointestinal tract. Piloerection was noted in 1/2 animal(s) at 500/1000 mg/kg bw/d.
At 1000 mg/kg bw/d an increase of water consumption (48% above control), a decrease of food consumption (16% below control), decrease of gross (20% below control) and corrected (30% below control) body weight gain was noted during treatment (GD 6-19). These animals transiently lost weight after initiation of treatment (GD 6-8). A dose of 500 mg/kg bw/d still caused a decrease of food consumption (12% below control) and a decrease of gross (13% below control) and corrected (14% below control) body weight gain.
In addition, at 500 and 1000 mg/kg bw/d the dams were affected by a regenerative hypochromic, macrocytic anemia, as was shown by decreased red blood cell (RBC) counts and mean corpuscular hemoglobin concentration (MCHC) as well as increased absolute reticulocyte counts and mean corpuscular volume (MCV). The anemia was accompanied by dose-dependently increased absolute/relative liver and kidney weights along with corresponding changes of clinical chemistry parameters. At 1000 mg/kg bw/d livers appeared already macroscopically enlarged.
Thus, dose levels of 50, 250 and 800 mg/kg bw/d were considered appropriate for the present definitive study, as the degree of the adverse effects should neither be potentially lethal nor should it cause severe suffering. Thus, the selected high dose for the present study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Di-rective 86/609/EEC on animal protection.
The conception rate was 96% in the low- and high-dose groups (50 and 800 mg/kg bw/d) and 100% in the control and the mid-dose groups (0 and 250 mg/kg bw/d).With these rates, a sufficient number of pregnant females were available for the purpose of this study according to the respective test guidelines).
The test substance, tested for its prenatal developmental toxicity, was administered as an oily preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 50, 250 and 800 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 (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 24-25 females per group had implantation sites.
Water consumption, 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, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including sampling of thyroid glands (with parathyroid glands) and weight determinations of the kidneys, liver, spleen, thyroid glands (with parathyroid glands), unopened uterus and placentas). 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. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.
All females of the high- and mid-dose groups (800 and 250 mg/kg bw/d) and 8 females of the low-dose group (50 mg/kg bw/d) showed transient salivation during the treatment period. Furthermore, all females of the high- and mid-dose groups and 12 females of the low-dose group ploughed nose-first into bedding. Both findings occurred in most of the respective animals only within the 2-hour examination interval immediately after treatment, however in some mid- and high-dose dams it continued beyond 2 hours for a maximum of 5-hours. They are 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. They are not considered to be signs of systemic toxicity.
For 19/25 high-dose females (800 mg/kg bw/d) unsteady gait shortly after treatment (i.e. 0-2h) was recorded on GD 7-14, the highest number of animals being affected on GD 9 and 10. Furthermore, all females of the high-dose group had piloerection during the treatment period. Both findings are considered as treatment-related signs of systemic maternal toxicity.
Generally, clinical observations revealed no effects of treatment-related systemic maternal toxicity in the animals receiving 50 mg/kg bw/d of the test substance.
The mean food consumption of the high-dose dams (800 mg/kg bw/d) was decreased statistically significantly on GD 6-13 (: up to -44% vs. ctrls.). Afterwards it recovered, but during the treatment period (GD 6-19) the high-dose dams consumed 14% less food in comparison to the concurrent control group. The high-dose of the test substance consistently affected the gross and corrected (net) body weight gain (body weight loss GD6-8) of the dams, gaining overall about 16% (gross) or 33% (net) less weight than the concurrent control during the treatment period (GD 6-19). The carcass weight was about 4% below the concurrent control group. These effects were regarded to be treatment-related and adverse.
Regarding clinical pathology, in high dose dams (800 mg/kg bw/d) a regenerative hypochromic, macrocytic anemia was observed because of decreased red blood cell (RBC) counts and mean corpuscular hemoglobin concentration (MCHC) as well as increased absolute reticulocyte counts, mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH). Lower inorganic phosphate and calcium levels in these individuals was found, but the reason for these findings cannot be elucidated. In mid dose dams (250 mg/kg bw/d), RBC counts and MCHC were lower and MCV and MCH higher compared to controls. However, all values in this test group were within historical control ranges. Thus, these alterations are likely precursory to a more distinct anemia, but still adaptive changes which are not yet adverse.
In mid and high dose dams (250 and 800 mg/kg bw/d) decreased T3 and T4 values and increased TSH values indicated a hypothyroidism.
Regarding pathology, the significant absolute and relative weight increase of the liver in the mid and high dose animals (250 and 800 mg/kg bw/d) was consistent with an increased hepatocytic accumulation of, most likely, glycogen. Histopathologically, this accumulation was similarly present in both test groups and was regarded as treatment-related but not as adverse since neither signs of cytotoxicity nor changes in clinical chemistry parameters indicative of hepatic alteration were noted.
In the thyroid glands, histopathology revealed a minimal hypertrophy/hyperplasia of the follicular cells in 2/24 high-dose dams (800 mg/kg bw/d). The minimal incidence and grading of these findings were assumed as possibly treatment-related and in combination with the results of hormonal measurements (increased TSH and decreased T3 and T4) as potentially adverse.
In high dose dams, the absolute and relative kidneys and spleen weights were significantly increased above the historical control ranges. In a previous study with a 28-day exposure to the same test substance, significant absolute and relative kidneys weight increases occurred in females at 300 and 1000 mg/kg bw/d without a histopathological correlate, whereas significant relative spleen weight increases at 1000 mg/kg bw/d were consistent with extramedullary hematopoiesis and hematological signs of regenerative anemia. The data of the 28-day study and the absence of altered clinical chemical markers of kidney toxicity in the present study indicate that the kidney weight increases were treatment-related but not adverse. Although no histopathological investigation of the spleen was performed in the present study, the significant weight increases of the organ in the high dose group were assumed to be a treatment-related adaptive response, relating to the evident adverse hematological signs of regenerative anemia.
No differences of toxicological relevance between the control and the treated groups (50, 250 or 800 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 post-implantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose.
The mean fetal weights of the mid- and high-dose groups (250 and 800 mg/kg bw/d) were statistically significantly reduced (about 5% and 11% below control). This correlated with mild delays in ossification of a few skeletal elements (skull, sternebrae, sacral arch). These delays of ossification do not constitute structural alterations of the skeleton, as the underlying cartilage template was completely intact in all these cases. Further, the delayed ossification noted in the mid- and high-dose group was associated with beginning (250 mg/kg bw/d) or distinct (800 mg/kg bw/d) maternal toxicity. As can be seen from the historical background data, increased incidences of such incomplete or non-ossifications of skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this Crl:WI(Han) rat strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. Thus, their toxicological relevance is considered to be rather low.
Under the conditions of this study the test item is not teratogenic.
In conclusion, 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) caused evidence of distinct maternal toxicity (such as adverse clinical findings, reductions of food consumption and body weight/body weight gain, anemia, thyroid perturbation at 800 mg/kg bw/d) under the conditions of this prenatal developmental toxicity study, Beginning maternal toxicity was detectable already at 250 mg/kg bw/d where clinical findings, precursory signs of anemia and thyroid perturbation were noted. Each of these effects was not strong enough to constitute adverse events when looked at them individually, but as a whole provide evidence for disturbance of maternal homeostasis. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg bw/d.
Further, the test item caused a slight delay of embryofetal development (lower fetal weights, mild delays in ossification) at dose levels of 250 and 800 mg/kg bw/d. These dose levels also caused maternal toxicity. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 50 mg/kg bw/d. However, test substance did not show any teratogenic potential under the conditions of this study.
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