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EC number: 270-128-1
CAS number: 68411-46-1
Information on reproductive toxicity is available from an OECD 422, as well as an OECD 443 study.
OECD 422:Combined 28-day repeated dose toxicity study with the reproductin/developmental toxicity screening test in rats by oral gavage:
Dosages: 25, 75, 225 mg/kg bw/d
Parental NOAEL: 25 mg/kg bw/day:
Hepatic toxicity in animals of both sexes at 225 mg/kg bw/day. At 75 mg/kg bw/d, morphologic liver findings were regarded to be an adaptive, non-adverse response. However, additional changes at 75 mg/kg bw/d in clinical biochemistry and a relative liver weight increase were considered toxicologically relevant.
Reproduction NOAEL: 225 mg/kg bw/day:
No reproductive toxicity was observed up to 225 mg/kg bw/day.
OECD 443: Extended One-Generation Reproduction Toxicity Study in Wistar Rats Administration via the Diet:
Dosages: 200, 600, 1800 ppm. With 10 weeks premating, extension of cohort 1B and developmental neurotoxicity cohorts 2A and 2B.
General, systemic toxicity LOAEL F0: approx. 18 mg/kg bw/d (200 ppm):
Evidence for liver toxicity and corresponding thyroid histopathology and thyroid hormone changes in all test groups
General, systemic toxicity NOAEL F1: approx. 18 mg/kg bw/d (200ppm):
Distinct toxicity such as decreased body weight/body weight gain, anemia as well as liver and thyroid toxicity.
Fertility and reproduction NOAEL in F0 and F1: Approx. 54 mg/kg bw/d (600ppm):
Lower numbers of implants and subsequently smaller litters.
No toxicologically relevant effects on reproductive parameters were
noted with treatment up to 225 mg/kg bw/day. The mating, fertility and
conception indices, precoital time, and number of corpora lutea and
implantation sites were unaffected by treatment.
There were 10, 10, 10 and 9 pregnant females in the control, 25, 75 and
225 mg/kg bw/day groups, respectively. For female nos. 46 (Group 1) and
55 (Group 2), the number of pups were slightly higher than the number of
implantations. This was considered caused by normal resorption of these
areas as these enumerations were performed on Day 5 or 7 of lactation.
No toxicologically relevant effects on the gestation index and duration,
parturition, maternal care or on most aspects of early postnatal pup
development (clinical signs, body weight and macroscopy) were observed
up to 225 mg/kg bw/day.
+/++ Steel-test significant at 5% (+) or 1% (++) level
Viability index = (Number of alive pups before planned necropsy / Number
of pups born alive) *100
# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level
Number of pups at first litter check and postnatal loss for the high
dose group (225 mg/kg bw)
Pup Bodyweights (g) during lactation
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1%
The test substance was tested for reproductive toxicity in an OECD 422, an OECD 421 'plus' range finding, as well as an OECD 443 study. All studies were performed in compliance with GLP regulations.
In the OECD 422 combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, rats were treated with the test substance by oral gavage at dose levels of 25, 75 and 225 mg/kg bw/d. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). Females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 39-45 days). In parental animals hepatic toxicity was noted for animals of both sexes at the high dose of 225 mg/kg bw/d. This was supported by changes in clinical pathology endpoints (increase of ALP, bilirubin, cholesterol and decrease of albumin, total protein and inorganic phosphate, among others), macroscopic abnormalities (accentuated lobular pattern, enlargement and discoloration), increased liver weights (relative weights approximately 50% higher for males) and microscopic findings including hepatocellular vacuolation and hypertrophy. At 75 mg/kg bw/day, morphologic liver findings were regarded to be an adaptive, non-adverse response based on the minimal severity of the hypertrophy (correlated to pale discoloration) and low incidence and minimal degree of vacuolation in the females. However, when taken together with similar changes seen in clinical biochemistry parameters in high dose animals and an approximate relative liver weight increase of 24% and 17% for males and females, respectively, the findings were also considered toxicologically relevant at 75 mg/kg bw/day. Organ weight changes at 25 mg/kg bw/day were not considered toxicologically relevant as there were no corroborative changes in clinical biochemistry parameters indicative of liver damage. No treatment-related toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption). Regarding reproductive toxicity, no toxicity was observed up to the highest dose level tested (225 mg/kg bw/day). Effects in developmental toxicity were seen as females at 225 mg/kg bw/day had a lower mean number of pups at the first litter check compared to controls (7.9 versus 10.9). An increase in postnatal loss and a correspondingly lower viability index were seen for females at this dose level (8 versus 0 in controls). While the majority of the postnatal loss was attributable to one female, other litters were also affected and a possible effect of treatment could not be excluded. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development including, clinical signs, body weight and macroscopy). In conclusion, the treatment of male and female Wistar Han rats by oral gavage at dose levels of 25, 75 and 225 mg/kg revealed parental (hepatic) toxicity at 75 and 225 mg/kg bw/day. Developmental toxicity was seen at 225 mg/kg bw/day only. No reproductive toxicity was observed with treatment up to 225 mg/kg bw/day.
The range-finding study (OECD 421 plus) was performed prior to the OECD 443 study, with a 10 week-premating period. Dietary test substance concentrations of 300, 1000 and 3000 ppm were fed to 10 Wistar rats per dose and sex (concentrations were reduced to 50% during lactation). After the acclimatization period, the test substance was administered as addition to the diet continuously throughout the entire study. The duration of treatment covered a 10-week premating in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 13 days of lactation and roughly 2 weeks thereafter in females. The animals of the control group were treated in the same way with the vehicle only (diet). Treatment ended about 16 - 20 hours before sacrifice. Ten weeks after the beginning of treatment, the surviving males and females from the same test group were mated overnight in a ratio of 1:1 for a maximum of 2 weeks. On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups. Signs of general systemic toxicity were observed in all parental animals of the high dose group (3000 ppm) and in females of the intermediate dose group (1500 ppm) which showed alterations of food consumption and body weight (changes). Clinical pathology indicated as changes in liver metabolism as decreased total bile acid (TBA) levels in rats of both sexes of the 1000 and 3000 ppm test groups and increased triglyceride values in male and female rats of the high dose group as well as decreased albumin and increased cholesterol values in females of the high dose group. Higher activities of gamma-glutamyl transferase (GGT) in dams of the high dose group and increased alkaline phosphatase (ALP) activities in both sexes of the 1000 and 3000 ppm groups as well as in females of the low dose group (300 ppm) were most probably due to a liver enzyme induction coupled with a liver cell swelling. Most probably in consequence of the liver enzyme induction, T4 values in adult high dose group males were decreased because of accelerated clearance of the conjugated hormone via the bile. As feedback mechanism TSH values were increased in these individuals. Enzyme induction led to lower total bilirubin levels in high dose group males because of an increased bilirubin conjugation and accelerated excretion via the bile. In contrast, in dams of this test group total bilirubin values were increased, presumably because of a bile congestion. Consistently, decreased total bile acid levels in rats of both sexes of intermediate and high dose groups (1000 and 3000 ppm) were most probably due to a decreased bile acid synthesis because of the changed liver cell metabolism. This was confirmed at least in high dose group females by lower albumin synthesis leading to lower serum levels of albumin. Pathological examination exhibited, the liver and the thyroid glands in males and females as target organs. The liver showed a significant absolute and relative weight increase in males and a significant relative weight increase in intermediate and high dose group females. A minimal but significant relative weight increase was also noted in low dose group males. These weight changes were consistent with liver cell hypertrophies and fatty changes, which showed varying patterns at the different dose levels. A significant relative weight increase of the thyroid glands in high dose group males correlated with histopathological and hormonal changes. In females, a significant final body weight decrease was observed in the intermediate and high dose groups (-9% and -15%, respectively). At 3000 ppm histopathology showed a hepatocellular hypertrophy in the liver which was mainly diffuse with mild centrilobular accentuation (minimal to moderate) affecting all male and female animals. A periportal fatty change, mainly of microvesicular with some macrovesicular type was observed in males (minimal) and females (minimal to slight). An additional focal necrosis was found in one male. All these changes associated with significant and relevant relative liver weight increases (males +43%, females +30%) were regarded as treatment related and adverse. In the thyroid glands of males, hypertrophy/hyperplasia of follicular cells showed a increase in incidence (9 out of 10 males) and grading (minimal to moderate) accompanied by altered, flaky colloid, also increased in incidence and grading. These changes were regarded as treatment-related and adverse as they were consistent with altered hormonal values in the clinical chemistry (decreased T4 and increased THS, both statistically significant). In females, the hypertrophy/hyperplasia of follicular cells, also accompanied by altered colloid with flaky appearance, showed a lesser incidence and grading than in males (6 out of 10, minimal to slight) but it was also considered treatment-related. However, since thyroid gland hormones were not altered, the histopathological findings were assessed as treatment related but not adverse. At the intermediate dose of 1000 ppm, all males showed centrilobular hepatocellular hypertrophy (minimal to slight), correlating with significant liver weight increases (absolute +12%, relative +14%). In addition, minimal fatty change (4 out of 10 males) of predominant macrovesicular type and midzone localization, together with minimal single cell necrosis/apoptosis (6 out of 10 males) and focal necrosis (one male out of 10) was observed. In females, 8 out of 10 showed a minimal centrilobular liver cell hypertrophy correlating with a significant relative weight increase (+16%). The thyroid glands of males showed minimal hypertrophy/hyperplasia of follicular cells (4 out of 10 males) accompanied by altered flaky colloid. These changes were assessed as treatment-related but not adverse due to the lack of hormonal changes. In females, a minimal hypertrophy/hyperplasia of follicular cells (2 out of 10), although no clearly dose-dependent, was associated with the presence of altered flaky colloid and was assumed to be possibly treatment-related but not adverse. In the low dose group (300 ppm), 2 out of 10 male animals displayed centrilobular hepatocellular hypertrophy (minimal and slight). Furthermore, minimal single cell necrosis/apoptosis was present in these two male animals. All these findings were considered treatment-related and potentially adverse. No treatment-related findings were observed in female animals. In the thyroid gland, the incidence of hypertrophy/hyperplasia of follicular cells in males (3 out of 10) and females (3 out of 10) were minimally above the historical control data and therefore, they were assumed as possibly treatment-related but not adverse. Effects on fertility and reproductive performance were seen in the high dose group (3000 ppm). A treatment related increase of estrous cycle length as well as decreased number of implantations sites and pups delivered. No signs of reproductive toxicity were observed in male or female parental animals of the 300, and 1000 ppm groups. Developmental toxicity was observed in the high dose group as decreased body weight gain was observed in male and female pups starting from after birth and resulting in decreased body weight from postnatal day (PND) 7 onwards. The deviations from control values were increasing over time. In these male pups the number of areola/nipples and the nipple number per animal were higher. Since the sexual maturation is related to the body weight development (Melching-Kollmuss et al 2017), this observation is at least partially related to the delay of general development in male pups. Furthermore, a significant lower number of pregnant rats was observed without dose-dependency in the lowest dose group (300 ppm), which was not assessed as treatment-related. Under the conditions of this Reproduction/Developmental Toxicity Screening Test in Wistar Rats, the oral administration of Benzenamine, N-phenyl-, reaction products with 2,4,4- trimethylpentene to male and female Wistar rats via the diet revealed signs of systemic toxicity at all concentrations tested from 300 to 3000 ppm. Based on altered liver parameters in clinical pathology and pathology, the LOAEL for general systemic toxicity is 300 ppm for male (26 mg/kg bw/d) and female Wistar rats (28 mg/kg bw/d). The NOAEL for reproductive performance and fertility is 1000 ppm for male (87 mg/kg bw/d) and female (95 mg/kg bw/d) Wistar rats. The NOAEL for developmental toxicity is 1000 ppm (95 mg/kg bw/d) in parental females.
In the OECD 443 guideline study, the test substance was administered to groups of 25 male and 25 female young Wistar rats as a homogeneous addition to the food in different concentrations (0, 200, 600 and 1800 ppm). F0 animals were treated at least for 10 weeks prior to mating to produce one litter (F1 generation). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 4 different cohorts (1A, 1B, 2A and 2B) which were subjected to specific postweaning examinations. Cohort 1B (=F1 generation parental animals) was selected to produce F2 pups. Cohort 2A and 2B animals were assigned as developmental neurotoxicity cohorts. Cohort 2A animals were sacrificed after 11 and cohort 2B animals after 3 weeks. F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were offered diets containing target dosages of 0, 200, 600 and 1800 ppm of the test substance post weaning, and the breeding program was repeated to produce a F2 litter. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1B. Test diets containing the test substance were offered continuously throughout the study. During the lactation period the test substance concentrations in the diet of the F0 and F1 females were reduced to 50%. The general state of health of parental animals and the pups was checked each day, and parental animals were examined for their mating and reproductive performances. Food consumption of the F0 parents and F1 rearing animals was determined regularly once per week (except for food consumption of the F0 and F1B males which was determined after the 10th premating week) and weekly for F0 and F1B females during gestation days (GD) 0-7, 7-14, 14-20 and postnatal days (PND) 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21. In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 and F1B females were weighed on GD 0, 7, 14 and 20 and on PND 1, 4, 7, 10, 14, 18 and 21. A detailed clinical observation was performed in all F0 parents and F1 animals in cohorts 1A, 1B and 2A at weekly intervals. Estrous cycle data were evaluated for F0 and F1B females over a three week period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. Moreover, the estrous stage of each F0, 1A and 1B female was determined on the day of scheduled sacrifice. The F1 and F2 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. At necropsy, all pups were examined macroscopically (including weight determinations of brain, spleen and thymus in one pup/sex/litter of F2 pups). Anogenital distance measurements were conducted in a blind randomized fashion on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one day prior to necropsy. The number of nipple/areola anlagen was counted. Date of sexual maturation, i.e. day of vaginal opening or balanopreputial separation of all F1 pups selected to become F1 rearing animals (except F2B rearing animals) was recorded. Urine samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Further blood samples were taken from all surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining. All F0 and F1B parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females of cohort 1A. All F1 rearing animals were assessed by different pathological, neuro- and histopathological examinations. Concentration and homogeneity analysis of treated feed verified the homogeneity and confirmed correct target concentrations of the test substance within the feed. The overall mean doses throughout all study phases and across all cohorts for males were approx. 18 mg/kg bw/d in the 200 ppm group, approx. 54 mg/kg bw/d in the 600 ppm group and approx. 167 mg/kg bw/d in the 1800 ppm group and for females approx. 18 mg/kg bw/d in the 200 ppm group, approx. 54 mg/kg bw/d in the 600 ppm group and approx. 166 mg/kg bw/d in the 1800 ppm group. None of the tested groups showed treatment related clinical signs or mortality. The body weight and body weight change of 1800 ppm female F0 rats was below control values during major parts of premating as well as gestation and lactation periods. Although male animals of this dose level showed frequent episodes of decreased body weight gain body weights of these animals were not affected. In F1 animals of both sexes treated with 1800 ppm, body weights and body weight change were consistently below the control throughout the in-life period. Particularly, high dose group F0 and F1B dams showed weight reductions during their gestation and lactation periods. F0 and F1 animals of the 200 and 600 ppm dose groups, also showed episodes of drecreased body weight gain predominantly in males but also some in females. Magnitude and frequency of these changes was generally lower than at 1800 ppm but it may still represent a treatment related effect. Food consumption in the 1800 ppm F0 and F1B dams food consumption was consistently reduced during gestation and lactation. Additionally, reductions were seen in the F1B females throughout premating period. In the 200 ppm and 600 ppm dose group F0 males and females as well as in F1 adolescents (Cohort 1A, 1B and 2A) of all dose groups, the food consumption remained either unchanged or only showed episodes of inconsistent changes.
In clinical pathology of high dose group parental F0 male and female rats decreased hemoglobin and hematocrit values indicated an anemia. In males higher reticulocyte counts were indicative of a regenerative anemia. In the liver an increased synthesis of coagulation factors resulted in a reduced prothrombin time, higher triglyceride and lower albumin levels in rats of both sexes as well as lower total protein values in males of this test group. In females, this was accompanied by increased activities of γ-glutamyl transferase (GGT) activities as well as higher globulin and cholesterol values. Higher alkaline phosphatase (ALP) activities in male and female rats receiving 1800 ppm were also due to liver enzyme induction. Furthermore, an increased alanine aminotransferase (ALT) in males of this test group was observed. The altered coagulation factor synthesis led to decreased platelet counts in females. Higher ALP activities and triglyceride values were already observed in male and female rats of the 600 ppm test group. In high dose group F1 cohort 1A rats (1800 ppm) similar clinical pathology changes were observed at study day 90. At the intermediate dose level of 600 ppm in F1A male and female rats increased ALP activities and lower albumin levels, with lower total protein and globulin levels in males were seen. Increases of ALP activities and decreases of albumin levels were observed already in low dose group females (200 ppm).
In pathology treatment-related changes were seen in the liver and subsequently in the thyroid in F0 and F1 males and females. In the F0 parental generation, intermediate and high dose group males and females (600 and 1800 ppm) showed increased liver weight which was caused by a diffuse or centrilobular hepatocellular liver cell hypertrophy and in males by an increase of fat in the midzonal-centrilobular region. In combination with changed parameters in clinical pathology these findings were considered as treatment-related and adverse. A minimal centrilobular fat storage in two male animals and a minimal hepatocellular centrilobular hypertrophy in seven females were already seen at the low dose which was regarded as treatment related but not adverse. F1 cohort 1A females of the intermediate and high dose groups (600 and 1800 ppm) showed increased liver weight which corresponded to a centrilobular liver cell hypertrophy. In the same test groups males revealed an increase in centrilobular hypertrophy and fatty change. In combination with findings in clinical pathology, the weight changes and liver cell hypertrophy were regarded to be treatment-related and adverse. In the F1 cohort 1A low dose group (200 ppm) two males revealed centrilobular hypertrophy. As clinical pathology parameters were not changed, this finding might be treatment-related but not as adverse. In F1 cohort 1B, high dose group male animals (1800 ppm) showed an increase in relative liver weight and females of the same test group an increase of absolute and relative weight. Other findings in this cohort occurred either individually or were biologically equally distributed over control and treatment groups.
In the thyroid glands of F0 males and females of all test groups hypertrophy/hyperplasia of follicular cells and altered colloid were observed, which corresponded to increased organ weights in the high dose group. Because there were also changes in hormone levels observed in clinical pathology, these findings were regarded to be treatment-related and adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. Regarding F1 cohort 1A animals, the thyroid glands of high dose group males and females of the low and high dose groups (600 and 1800 ppm) showed follicular cell hypertrophy/hyperplasia. Altered colloid was only seen in male and female animals of the high dose group (1800 ppm). In combination with clinical pathology findings in these test groups, the finding was regarded to be treatment-related and adverse. Because further findings in the thyroid glands of intermediate and low dose group males did not show a dose-response relationship and lacked findings in clinical pathology they were considered as incidental and unrelated to treatment.
Measurement of T4 and TSH thyroid hormones revealed effects in the F0 and F1 adult animals. No effects were observed in the F1 and F2 preweaning offspring. In F0 parental rats, T4 decreases were observed in males and TSH increases were found in both sexes of all dose groups. Regarding the findings made in liver parameters, it can be assumed that the thyroid hormone level changes happened secondary to a liver cell enzyme induction because of an accelerated clearance of T4 via the liver coupled with an enhanced T4 synthesis induced by higher TSH releases via an endocrine feedback mechanism. It also seems that this feedback maintains the serum T4 homeostasis in parental females, but not in F0 males. In high dose group F1 cohort 1A animals of both sexes TSH values were increased, but T4 values were decreased only in males of this test group. This corresponds to the thyroid hormone changes mentioned in F0 rats. The mechanism of increased clearance of T4 via the liver is supported by the fact that exclusively the grown-up F1 rats showed thyroid hormone changes, because liver cell enzyme activity may not be fully established in the pups until PND 22. In F1A females of the intermediate dose group (600 ppm) T4 values were significantly decreased and TSH values were increased. Although T4 decreases were not dose dependent, these findings correlate with the histopathological observations in the thyroids.
There were no indications from clinical examinations as well as gross and histopathology, that treatment adversely affected the fertility or reproductive performance of the F0 and F1B parental animals up to and including the administered intermediate dose of 600 ppm. Estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and gross and histopathological findings of these organs (specifically the differential ovarian follicle count) were comparable between the rats of low- and mid-dose and control groups and ranged within the historical control data of the test facility.
At a dose of 1800 ppm significantly reduced numbers of implants were observed, which were below the range of historical controls. This subsequently caused a smaller average litter size in F1 and F2 offspring of the high dose group. However, because estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and morphology of these organs were unchanged in the F0 and F1B parents of this offspring, it is likely that the considerable stress caused by maternal toxicity at the high dose, which was particularly distinct during gestation/lactation, is related to the lower implantation success.
Pup body weight development was affected in high dose group offspring (1800 ppm) approximately from day 7 of lactation. The offspring weighed less than controls during the course of lactation and their weights were below the historical control range. There was no treatment-related influence on high dose F1 and F2 pup body weight change at birth and shortly thereafter. In the last two weeks of lactation (PND 7 - 21) the mean body weight change of these pups was significantly below control values. The F1 intermediate dose offspring (600 ppm) was similarly affected, but to a lower extent. The F2 intermediate dose level offspring remained unaffected. During the last week of lactation the offspring already consumed considerable amounts of treated diet and the post-weaning body weight gain of the high dose adolescents in the various F1 cohorts continued to be lower compared to the controls. Therefore, it was assumed that the lower pre-weaning body weight/body weight gain at 600 and 1800 ppm was caused by direct exposure of the offspring to the test compound through the diet as much as by developmental toxicity. However, the pup body weight effects had no influence on postnatal pup survival or well-being, neither during early lactation nor later, as clinical and/or gross necropsy examinations of the mid and high dose F1 and F2 pups revealed no adverse findings.
Analysis of the anogenital distance/index and the presence of nipples/areolas revealed no findings, indicating the absence of endocrine-mediated imbalances. However, there was no difference in the percentage of F2 male pups having nipples/areolae but the mean nipple number per pup was statistically significantly above the concurrent control values in the 1800 ppm F2 male pups when examined on PND 13. It should be noted thtat this is considered to be the consequence of a general delay of pup development, rather than a specific effect on hormonal homeostasis. Furthermore, no nipples/areolae were detected in any male pup of all test groups during the re-examination on PND 20.
Regarding developmental landmarks, a statistically significant delay in vaginal opening of less than one day beyond the concurrent control was observed in female F1 offspring of the high dose group (1800 ppm). However, the delayis within the historical control range of the test facility and there was a consistent effect on postweaning body weight development in the affected females notable, and no corresponding increase in body weight was observed in the older animals at the time of puberty. Furthermore, there was no effect on estrous cyclicity or the integrity of sexual organs in these females, including differential ovarian follicle count, during later life. Similarly, there was a statistically significant delay in preputial separation of about 1.5 days, which exceeded the control data in male F1 offspring of the high dose group. The delay is within the historical control range of the test facility. Again, there was a consistent effect on postweaning body weight development in the affected males notable and no corresponding increase in body weight was observed in the older animals at the time of puberty. In addition, no effect on the integrity of sexual organs (including accessory sexual glands) was noted in all treated males. Hence, it was assumed that this rather small delay in onset of puberty in both sexes was most likely a consequence of systemic toxicity and subsequent general developmental delay, and not a specific effect on the timing of puberty. Furthermore, there was no evidence that the test substance impaired development of neuronal function in the F1 offspring as demonstrated by the absence of relevant effects in a functional observation battery (FOB) as well as automated motor activity and auditory startle. However, in neurohistopathology of cohort 2A animals, which were sacrificed at PND 77 (includes post-weaning), an increased incidence of focal to multifocal axonal degeneration in the white matter characterized by digestion chambers with occasional pyknotic nuclei and presence of gitter cells was observed in male animals of the high dose group. No corresponding analysis for this specific finding was performed in cohort 2B animals, which were sacrificed on PND 22. Because it is not possible based on this data to differentiate wether this effect is a chronic toxicity effect or rather a developmental toxicity effect, the respective analysis of cohort 2B (PND 22) animals is scheduled for analysis.
Overall, the NOAEL for general, systemic toxicity in this study is below 200ppm (about 18 mg/kg bw/d) in the F0 parental rat, based on evidence for liver toxicity and corresponding secondary thyroid histopathology and thyroid hormone changes in all test groups. In F1 adult rats, the NOAEL is 200 ppm. At 1800 ppm (about 167 mg/kg bw/d) distinct toxicity such as decreased body weight/body weight gain, anemia as well as liver and subsequent thyroid toxicity was noted in the F0 parental animals as well as adolescent and adult F1 offspring, includingF1B parental rats.The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 600 ppm (about 54 mg/kg bw/d), based on lower numbers of implants and subsequently smaller litters, in the presence of maternal toxicity.
The NOAEL for developmental toxicity in the F1 and F2 progeny is 200 ppm (about 18 mg/kg bw/d), based on reduced preweaning body weight gain, which was presumably caused by direct exposure of the offspring to the test compound through the diet as much as by developmental toxicity.
Regarding findings in the spinal cord of cohort 2A animals, further tests have been scheduled to be able to make a final conclusion wether the findings in neurohistopathology should be regarded as developmental effects or rather as chronic toxicity effects.
Information on developmental toxicity is available from an OECD 422, as well as an OECD 443 study.
Developmental NOAEL: 75 mg/kg bw/day:
Females at 225 mg/kg bw/day had a lower mean number of pups at the first litter check. An increase in postnatal loss and a correspondingly lower viability indexwere seen for females at this dose level.
Develeopmental toxicity NOAEL in F1 and F2: Approx. 18 mg/kg bw/d (200ppm):
Reduced preweaning body weight gain.
Develeopmental neurotoxicity NOAEL in F1: Approx. 54 mg/kg bw/d (200ppm):
Increased incidence of focal to multifocal axonal degeneration in the white matter of thoracic cord. However, no clear distinction can be made wether this effect is a developmental or a chronic toxicity effect. Therefore, further analysis has already been initialized.
In the control and high dose group, each one dam was not pregnant. Mean
values were only calculated for pregnant animals.
Table 1: TERMINAL BODY WEIGHT, UTERUS WEIGHT, CORRECTED BODY WEIGHT AND
CORRECTED BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA
* = Statistically significantly different from control group value at p<
Table 2: FOOD CONSUMPTION (g/animal/day) - GROUP MEAN
** = mean value of group is significantly different from control at p <
Table 3: Pregnancy status overview
Table 4: Litter data and sex ratios - group mean data
Table 1: Excerpt of the results of maternal clinical signs during
Day of gestation
# OF FEMALES EXAMINED
BLOOD IN BEDDING BEFORE
ABORTION AFTER TREATMENT
ABORTION BEFORE TREATMENT
REDUCED DEFECATION BEFORE
REDUCED DEFECATION AFTER
NO DEFECATION BEFORE
NO DEFECATION AFTER
Table 2: Summary of reproduction data
TEST GROUP 0
TEST GROUP 1
TEST GROUP 2
TEST GROUP 3
Females Mated (N)
Conception Rate (%)
Premature Births (N)
Dams with Viable Fetuses (N)
Dams with all Resorptions (N)
Female Mortality (N)
Pregnant at Terminal Sacrifice (N)
Corpora Lutea MEAN
Implantation Sites MEAN
Preimplantation Loss MEAN (%)
Postimplantation Loss MEAN (%)
Dunnett-test (two-sided), *
: p<=0.05 ** : p<=0.01; Fi
=Fisher's exact test (one-sided)
3: Mean maternal body weights during gestation (g)
0 mg/kg bw/d
10 mg/kg bw/d
30 mg/kg bw/d
100 mg/kg bw/d
D=Dunnett-test (two-sided), *
: p<=0.05 ** : p<=0.01
4: Mean maternal body weight change during gestation (g)
D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01
5: Mean maternal food consumption during gestation
0 TO 6
MEAN OF MEANS
6 TO 28
0 TO 29
Table 6: Summary of maternal necropsy observations
NOTHING ABNORMAL DETECTED
FINDINGS AFTER GAVAGE ERROR
LUNGS: ACUTE FIBRINOUS -
LIVER: GRANULATED SURFACE
RECTUM: NO FECES
Table 7: Summary of reproduction data of the females
TEST GROUP 0
Resorptions: Total MEAN
Dead Fetuses (N)
D=Dunnett-test (two-sided), *: p<=0.05 ** : p<=0.01
8: Mean placental and fetal body weights (based on litter)
PLACENTAL WEIGHTS UNITS; GRAMS:
of Male Fetuses
of Female Fetuses
FETAL WEIGHTS, UNITS:
of all Viable Fetuses
Total fetal external malformations
Test group 0
Test group 1
Test group 2
Test group 3
bw/d = milligram per kilogram body weight per day; N = number; % = per
10: Total soft tissue malformations
11: Total skeletal malformations
Table 12: Fetuses with more than one malformation
Doe No.-Fetus No., Sex
0 (0 mg/kg bw/d)
domed head, hydrocephaly
1 (10 mg/kg bw/d)
thoracic hemivertebra, misshapen thoracic vertebra
malpositioned kidney, short ureter
2 (30 mg/kg bw/d)
exoccipital fused with 1st cervical arch, cervical hemivertebra
multiple malformations of the great vessels (persistent truncus arteriosus, aortic arch atresia, malpositioned subclavian origin)
aortic arch atresia, malpositioned kidney
thoracic hemivertebra, branched rib
3 (100 mg/kg bw/d)
multiple external malformations (domed head, cleft palate, small tongue), hydrocephaly
multiple external malformations (domed head, cleft
palate, small tongue)
palate, small tongue), severely malformed skull bones
bw/d = milligram per kilogram body weight per day; No.=
number; M = male; F = female
The stability of the test substance in 0.5% CMC suspension in deionized
water (with 10 mg/100 mL Cremophor EL) over a maximum of 7 days and
warmed up to approximately 30 degrees Celsius was demonstrated. The
homogeneous distribution of the test substance in the vehicle was shown.
The correctness of the prepared concentrations was shown.
Information on developmental toxicity from is available from an OECD 422 screening study, an OECD 421 'plus' dose range finding study, as well as an OECD 443 extended one-generation study.
For detailed study information please refer to additional information in the chapter effect on fertility.
The available experimental data does meet the criteria laid out for classification and labelling in EC Regulation 1272/2008. As a result the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).
Overall, lower numbers of implants with subsequently smaller litter sizes were likely caused by considerable stress through maternal toxicity at 1800 ppm, which was particularly distinct during gestation/lactation. Furthermore, no changes were seen in estrous cycle data, sperm quality, mating behavior, conception, gestation, parturition, lactation, and weaning as well as sexual organ weights and morphology.
Regarding developmental toxicity, lower pup body weights were attributed to direct exposure of the offspring to the test compound, resulting in a general delay of development. Furthermore, pup body weight effects had no influence on postnatal pup survival or well-being. Intermediate and high dose F1 and F2 pups revealed no adverse findings during early lactation or later, in clinical and/or gross necropsy examinations. Significant delays in vaginal opening and preputial separation in F1 offspring animals (which were however within HCD) were regarded as most likely a consequence of systemic toxicity and subsequent general developmental delay.
Histopathological findings in thyroids and thyroid hormone changes were seen as a consequence of liver cell enzyme induction and no direct effect of the test substance on the hormonal system.
For more details refer to the part additional information.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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