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Key value for chemical safety assessment

Effects on fertility

Description of key information

Two-Generation Toxicity Study (OECD 416), rats:

NOAEL general toxicity = 30 ppm (equivalent to 2.50 and 3.09 mg/kg bw/day in P0 males and females, respectively and equivalent to 3.68 and 4.18 mg/kg bw/day in P1 males and females, respectively)

NOAEL reproductive toxicity >= 3000 ppm (highest dose tested) (equivalent to 272 and 346 mg/kg bw/day in P0 males and females, repsectively and to 354 and 393 mg/kg bw/day in P1 males and females, repectively)

NOAEL offspring = 300 ppm (corresponding to 22.25 mg/kg bw/day)

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
272 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reliable studies on toxicity to reproduction toxicity are available.

As in other repeated dose toxicity studies with 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone in rats, corneal opacities were noted in the 2-generation reproduction study described below. Corneal opacities, occasionally accompanied by neovascularization and their histopathological correlates (keratitis, reactive epithelial hyperplasia, and vascularization) are considered a rat-specific phenomenon. Corneal changes were not seen in other species chronically treated with the test substance (i.e. mice and dogs). The test substance is an inhibitor of the HPPDase enzyme and induces increased plasma tyrosine levels. This effect is more pronounced in rats than in mice and dogs. Experimentally induced hypertyrosinemia has been shown to induce snow flake-like corneal lesions in rats but not in mice (M-210983-01-2). In mice and humans, even under conditions of strong HPPD inhibition, tyrosine concentrations will not increase to levels high enough to induce ocular toxicity and hence, this toxicity observed in the rat is inappropriate for extrapolation to humans (ECETOC TR No. 99, 2006).

Furthermore, and also as in other repeated dose toxicity studies with 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone in rats, thyroid findings were noted (increased weight, histopathological changes comprising changes in colloid, follicular cell hypertrophy and pigment deposition in the follicular epithelium) in the 2-generation reproduction study described below. These findings are considered a non-adverse and rat specific phenomenon.

No changes of the thyroid were noted in either mice or dogs, the other two species in which repeated-dose studies with histopathological examination of the thyroid were conducted with the test substance. This suggests that potential thyroid effects of the test substance have only a limited toxicological significance, as the rat is known to be much more sensitive than either mice or dogs to alterations in thyroid homeostasis. The main function of the thyroid is to regulate overall metabolic processes through synthesis, storage, and release of thyroid hormones. These hormones, thyroxine and triiodothyronine (T4 and T3 respectively) are produced by the action of thyroid peroxidase which iodinates tyrosine residues available in the thyreoglobulin complexes in the colloid of the thyroid follicles.

As the test substance through inhibition of the HPPDase enzyme increases plasma tyrosine concentration in the rat, it is quite possible that some of this increased tyrosine is taken up by the thyroid and stored in the colloid, either as free tyrosine or through either increasing the synthesis of thyreoglobulin or altering its composition in terms of number of tyrosine residues per thyreoglobulin molecule. As plasma tyrosine concentrations increase to a much lesser extent after HPPDase inhibition in the mouse than in the rat, this would explain why colloid alteration is not observed in the mouse even after administration of doses of up to approximately 600 mg/kg bw/day.

If alteration in the appearance of the thyroid colloid had an effect on the function of the thyroid, such an effect might be manifested in altered maintenance of body weight through alteration of metabolic rate, through alteration in the ability of the dams to maintain gestation, or through effects on offspring performance in the developmental neurotoxicity study.

In the rat 90-day, multigeneration, and chronic / oncogenicity studies, the alteration in thyroid colloid is seen at a lower dietary concentration of the test substance than is any effect on body weight or body weight gain. Additionally, the effect on offspring viability and body weight at parturition and during lactation is seen a greater dose than that in which thyroid colloid alteration is seen in the parental animals. These findings suggest that the altered colloid has no effect on thyroid function with regard to maintenance of normal body weight and development.

In the developmental neurotoxicity study, there was no biologically significant effect on any facet of learning or memory in the offspring at any dose. The ability of the offspring to master the water maze was unaffected. Although the latency to crossing in the passive avoidance test was slightly decreased at the high dose in both males and females, in the absence of other effects on learning and memory such a slight effect does not indicate that there was any mental impairment of the pups.

Histopathological sections of the thyroid from male and female rats from the rat chronic / oncogenicity study were examined by an independent panel of senior pathologists to determine whether the colloid alteration in the thyroid represented an adverse effect of the test substance. This expert group noted that the colloid alterations were present in all groups including controls, and that the morphology was similar between treated and control groups, with the primary difference being an increase in the number of follicles affected in treated groups. Additionally, colloid alterations were observed in the absence of follicular cell hypertrophy, and were not considered to indicate a persistent alteration in thyroid function.

From the observed and implied lack of functional consequences of the thyroid colloid alteration, as observed in multiple studies conducted with the test substance in the rat, it is clear that the finding of colloid alteration is of no toxicological significance and can be expected to be non-adverse.

 

Two generation reproductive toxicity study in the rat

In this study - that was performed under GLP and according to OECD 416 (M-263320-01-2) - the test substance was incorporated into rodent diet at concentrations of 0, 30, 300, and 3000 ppm and fed to male and female Wistar rats in the P0 generation throughout premating, mating, gestation, and lactation periods. Following weaning of the F1 generation, the weanlings were maintained in their same dietary groups through adolescence, mating, gestation, and lactation. The dietary concentrations used provided doses, during the pre-mating phase, of 0, 2.5, 26, and 272 mg/kg bw/day for male P0, 0, 3.7, 34, and 354 mg/kg bw/day for male F1, 0, 3.1, 33, and 345 mg/kg bw/day for female P0, and 0, 4.2, 39, and 393 mg/kg bw/day for female F1 animals.

Parental Findings

There was no treatment-related increase in mortality in either the P0 or the P1 males or females at any dose level. Two P1 females at 3000 ppm were sacrificed in a moribund condition, but their deaths were not considered to be related to treatment. The only treatment-related clinical sign was increased incidence of eye opacities in P0 and P1 animals at 3000 ppm during pre-mating, gestation, and lactation phases. During ophthalmological examinations the incidence of either diffuse or reticulate corneal opacities and of corneal neovascularization was increased in both P0 and P1 males and females at 300 and 3000 ppm. There was no treatment-related effect on body weight in the P0 males or females. In the P1 adults, body weight and body weight gain were statistically significantly reduced in males at 300 and 3000 ppm, and body weight was statistically significantly reduced in females at 3000 ppm, for a final reduction in body weight at 3000 ppm at the end of the pre-mating period of 12% in males and 7% in females. There was no statistically significant effect on body weight at any dose during gestation in either the P0 or the P1 dams. During lactation days 0-4, body weight gain of P0 dams at 3000 ppm and P1 dams at 300 and 3000 ppm was statistically significantly reduced compared to controls. These effects on body weight were considered to be treatment-related. There was no effect on food consumption during the pre-mating period in either male or female rats in the P0 or the P1 generation. Food consumption during lactation days 0-4 was slightly but statistically non-significantly reduced at 3000 ppm in P0 dams and at 300 and 3000 ppm in P1 dams. This effect on food consumption was evaluated as related to treatment.

There was no treatment-related effect on either estrus cyclicity or on any sperm analysis parameters in either the P0 or the P1 females or males, respectively. There were no treatment-related effects on any index of reproductive performance in either the P0 or the P1 adults. Both gestation and rearing indices were slightly decreased at 3000 ppm in the P1 adults, but these decreases were due to deaths not related to administration of the test substance, and therefore were themselves not related to treatment.

In P0 males, absolute kidney and liver weight were increased at 3000 ppm, and absolute thyroid weight was increased at 300 and 3000 ppm. Relative kidney, liver, and thyroid weights were increased in P0 males at 300 and 3000 ppm. There were slight increases in P0 females at 300 and 3000 ppm in absolute and relative kidney and liver weights. In P1 males, there were increases in absolute and relative liver, kidney, and thyroid weights at all doses which showed no relationship to dose. In P1 females, a dose-related increase in the absolute weight of liver and adrenals was seen only at 3000 ppm. Relative kidney, liver, and adrenal weight was increased in females at 3000 ppm. There were no treatment-related macroscopic findings in P0 male or female rats at any dose. In P1 males, one rat at 300 ppm and 14 males at 3000 ppm showed eye opacities. Two P1 females at 3000 ppm also showed eye opacities. There were no other treatment-related findings in P1 male or female rats.

There were no treatment-related effects on organs of the reproductive tract.

Treatment-related effects were observed in eyes and thyroid of both males and females of the P0 and P1 generations, and in pituitary, kidneys, and liver of P0 and P1 males only. Findings in the eyes, at 300 and 3000 ppm in both males and females of the P0 and P1 generations, included keratitis and vascularization, correlating with in-life observations. Thyroid findings included colloid alteration at all doses in male P0 and P1 animals and females of the P0 generations and from 300 ppm in P1 females, pigment deposition at all doses in P0 and P1 males and from 300 ppm in P0 and P1 females, and increased follicular cell hypertrophy in P0 males at 3000 ppm and P1 males from 3000 ppm and P0 females at 3000 ppm. Eosinophilic inclusions were increased in the anterior pituitary of males at 300 and 3000 ppm in both the P0 and P1 generations. Findings in the liver included hepatocellular hypertrophy, cellular alteration, and slight increases in periportal fat accumulation in P0 and P1 males at 300 and 3000 ppm. The kidneys of males in the P0 and P1 generation had increased incidence and / or severity of basophilic tubules and tubular dilation, in a dose-related manner from 300 ppm onwards.

Offspring findings

Dietary administration of the test substance did not affect number of implantations or prenatal loss. There was no effect on live birth index or on the ratio of males to females at any dose level. Viability indices were slightly, statistically non-significantly reduced in F2 pups at all doses. However, these decreases were considered not to be treatment-related through 300 ppm, as they were generally within the historical control range and at 30 ppm due to total litter loss of two dams before day 4 of lactation. There was no effect on ano-genital distance in either males or females in the F2 generation. The only treatment-related clinical sign in pups was the finding of animals cold to the touch, which was increased in F1 pups at 3000 ppm only (12 pups) compared to control (5 pups). There were no treatment-related findings in F2 pups. Ophthalmological examination of pups at the time of weaning on postnatal day 28 showed an increased incidence of corneal opacity and / or corneal neovascularization at 300 and 3000 ppm in both F1 and F2 pups, with a dose-related increase. Both litter (mean body weight for males and females taken together) and pup (mean body weight for males or females, taken separately) body weights were statistically significantly decreased in the F1 and F2 pups at 3000 ppm at weaning on day 28. In F2 females at 3000 ppm, body weight was also statistically significantly decreased at lactation day 21. These effects on pup and litter body weight were considered to be treatment-related. Treatment-related, statistically significant delays of preputial separation at 300 and 3000 ppm, and marginal delays of vaginal opening at 3000 ppm, were observed in the F1 pups.

There were no treatment-related effects on organ weights in either F1 or F2 weanlings. Absolute brain weight was statistically significantly decreased in male and female F1 and F2 weanlings. However, relative brain weight was unaffected, and thus the decrease in absolute brain weight was considered to be due to decreased body weight. Absolute spleen weights were statistically significantly decreased in F1 male and female weanlings at 3000 ppm, and in males relative spleen weight was also slightly but statistically significantly decreased. However, this effect was not observed in the F2 weanlings and thus was considered not to be treatment-related. Macroscopic findings: Corresponding with the increased mortality during lactation at 3000 ppm in the F2 pups compared to controls, treatment-related findings in this group at gross necropsy included increased incidence of autolysis and no milk in the stomach. The incidence of dilated and / or enlarged kidneys was increased at 3000 ppm in F1 and F2 pups.

Conclusions

Parental body weight was statistically and biologically significantly decreased by administration of high doses of the test substance by dietary incorporation. Treatment-related clinical signs in adult animals were limited to an increased incidence of eye opacity in both males and females. Liver, kidney and thyroid weights were increased, as well as the incidence of microscopic findings. In the liver, the microscopic findings were indicative of an adaptive response and of increased metabolic activity. Kidney findings were generally characterized as the result of an enhanced aging progress, and may be due to either excretion of the test substance in the urine, or to increased excretion of tyrosine in the urine due to HPPDase inhibition. The findings in the thyroid at 30 ppm were considered to be non-adverse in the absence of either functional changes or increase in other microscopic findings at this dose. The NOAEL for parental systemic toxicity was therefore 30 ppm (2.50 mg/kg bw/day in males, 3.09 mg/kg bw/day in females).

There was no effect on either sperm parameters or on estrus cyclicity at any dose, nor were there any effects on fertility, gestation index or length, or rearing indices. The reproductive NOAEL was therefore 3000 ppm (272.42 mg/kg bw/day in males, 345.67 mg/kg bw/day in females).

In pups, viability was reduced at 3000 ppm, and clinical signs were also increased in this dietary group. Pup and litter weights were reduced at 3000 ppm near the end of lactation, but there was no effect on pup body weight at birth. Preputial separation and vaginal opening were delayed with a greater effect on preputial separation. At necropsy, the incidence of dilated and/or enlarged kidneys was increased at 3000 ppm. The offspring NOAEL was therefore 300 ppm (22.25 mg/kg bw/day).

 

Effects on developmental toxicity

Description of key information

Developmental toxicity study (OECD 414), rats:

NOAEL maternal animals: 10 mg/kg bw/day

NOAEL fetuses: 10 mg/kg bw/day

NOAEL fetal abnormalities: 300 mg/kg bw/day (highest dose tested)

Developmental toxicity study (OECD 414), rabbits:

NOAEL maternal animals: 75 mg/kg bw/day

NOAEL fetuses: 10 mg/kg bw/day

NOAEL fetal abnormalities: 250 mg/kg bw/day (highest dose tested)

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies (Klimisch score 1), and are thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Toxicity to reproduction: other studies

Additional information

Reliable studies on developmental toxicity / teratogenicity are available in rats and rabbits.

 

Developmental toxicity study in the rat

In this study - that was performed under GLP and according to OECD 414 (M-267036-01-2) - the test substance was administered to groups of 25 female Sprague Dawley rats at doses of 10, 100, and 300 mg/kg bw/day on gestation days 6 through 20. On gestation day 21, the rats were sacrificed subjected to macroscopic examination. The number of ribs was counted and liver weight was measured. Gravid uterine weight was measured, and the number of corpora lutea, implantations, resorptions, and live and dead fetuses, and the sex and individual body weights of live fetuses were determined. Live fetuses were killed by subcutaneous injection of sodium pentobarbital and examined for external observations. Approximately half of the fetuses from each litter were preserved for free-hand sectioning, while the other half were skinned, eviscerated, and stained in alizarin red S and alcian blue. Fetal observations are classified as common variants (changes occurring in more than approximately 5% of the control population), minor anomalies (slight, relatively rare structural changes that are not obviously detrimental), and malformations (very rare or obviously lethal changes).

There were no treatment-related mortalities during the study. Treatment-related clinical signs were observed at 100 and 300 mg/kg bw/day, and included increased salivation, anogenital soiling, intense yellow urine, and vaginal discharge. There was no effect of treatment on pregnancy rate in any group. At 300 mg/kg bw/day, maternal body weight was slightly, and statistically not significantly, decreased during the latter part of gestation. Body weight change was statistically significantly decreased at 100 and 300 mg/kg bw/day in the interval of gestation days 6-8. Thereafter, body weight change was similar across all groups. Maternal corrected body weight change was statistically significantly reduced at 300 mg/kg bw/day. There was a treatment-related, biologically and/or statistically significant decrease in food consumption at 300 mg/kg bw/day between gestation days 6 and 16, compared to controls. The only treatment-related finding at gross necropsy was observed in one female at 300 mg/kg bw/day, which showed yellow sediment in the kidney and gritty content in the urinary bladder. Liver weight was statistically significantly increased in 300 mg/kg bw/day (15.6 g, p<0.05) and slightly increased at 100 mg/kg bw/day (15.4 g, not statistically significant) versus liver weight in control rats (14.7 g).

There were no treatment-related effects on the number of corpora lutea, implantation sites, either early or late resorptions, number of live fetuses, male/female ratio, or percent pre- or post-implantation loss per litter. Fetal body weight was biologically and/or statistically significantly decreased at 100 and 300 mg/kg bw/day.

Fetal external observations: A total of four fetuses at 300 mg/kg bw/day, from 2 different litters, presented the malformation of hindpaw polydactyly. The low incidence of this finding, the lack of other related findings in either forepaws or hindpaws, and the lack of ossification of the extra phalanges in the 2 fetuses of these 4 which were examined by skeletal staining suggest that this malformation was due to chance and was not an indication of a teratogenic potential of the test substance. One fetus at 10 mg/kg bw/day had the malformation of umbilical hernia, which was evaluated as not related to treatment. Three of the fetuses with hindpaw polydactyly (300 mg/kg bw/day), all of which were in the same litter, also showed an anomaly of forelimb hyperflexion, misshapen digits on the forepaws, and malpositioning of the digits on both the forepaws and the hindpaws. There were no other treatment-related anomalies or variants in any dose group. Examination of the fetuses for visceral findings revealed no malformations, and no treatment-related variants or anomalies, in any group. Fetal skeletal observations: Two of the four fetuses with the external observation of hindpaw polydactyly had malformations of short tibia and femur, supernumerary cartilaginous phalanges, fused cartilage for several metatarsals and phalanges, and unossified 5th metatarsal. These findings were considered not to be related to treatment, because of their low incidence. The only anomaly which was evaluated as related to treatment was an increase at 300 mg/kg bw/day of 27 pre-sacral vertebrae.

Treatment-related variants observed in this study were primarily linked to altered? ossification.

Conclusions

Based on decreased maternal body weight gain between gestation days 6 and 8, as well as clinical signs observed at 100 and 300 mg/kg bw/day, the maternal NOAEL in this study was considered to be 10 mg/kg bw/day. The fetal effects observed included decreased fetal body weight and delays in ossification, and therefore the NOAEL was 10 mg/kg bw/day.

 

Developmental toxicity study in the rabbit

In this study - that was performed under GLP and according to OECD 414 (M-266702-01-2) - the test item was administered by oral gavage to groups of 25 pregnant female New Zealand white rabbits at doses of 0, 10, 75, and 250 mg/kg bw/day from gestations days 6 through 28, inclusive. On day 29 of gestation, the rabbits were sacrificed and subjected to macroscopic examination. Liver weight and the number of ribs were noted for each animal. The gravid uterine weight, number of corpora lutea, number of implantations, resorptions, and live and dead fetuses, and the individual weight of live fetuses, were recorded for each animal. Live fetuses were sacrificed and examined externally. The heads of approximately half the fetuses were fixed for later examination. All fetuses were dissected for visceral findings, fixed, and stained for skeletal examination. Fetal observations were classified as common variants (changes occurring in more than approximately 5% of the control population), minor anomalies (slight, relatively rare structural changes that are not obviously detrimental), and malformations (very rare or obviously lethal changes).

There were no mortalities during the study. Yellow or beige sediments in the urine of females at 75 and 250 mg/kg bw/day were noted on several occasions, with the incidence greater at 250 mg/kg bw/day. The test substance is excreted in the urine and forms a yellow sediment, thus although this sediment was clearly treatment-related it was seen as an indication of compound excretion rather than as an adverse effect. There was no effect on pregnancy rate, with rates ranging from 88% to 96%. Maternal body weight gain at 250 mg/kg bw/day was statistically significantly reduced between days 8 and 10, with an overall reduction between days 6 and 29 of 31% compared to controls. Corrected maternal body weight at 250 mg/kg bw/day was slightly but statistically non-significantly decreased compared to controls. At 250 mg/kg bw/day, food consumption was consistently reduced compared to controls, with the reduction achieving statistical significance on several occasions. Liver weight was statistically significantly increased at 250 mg/kg bw/day (125.3 g, p ≤ 0.01) compared to controls (102.6 g). Prominent lobulation of the liver was noted at 250 mg/kg bw/day in 2 out of 25 females. Counting ribs in adult female rabbits revealed that the most common number of ribs was 26 (58% to 78% of dams per treatment group, 70% of all animals). As rib count is established prior to birth, this cannot be due to administration of the test substance. Instead, this data shows that rib count in rabbits can vary widely among groups, but that the general tendency is toward 26 ribs. At 250 mg/kg bw/day, fetal body weight was statistically significantly decreased both for combined sexes and for the sexes taken separately. This decrease was considered to be treatment-related. Although combined-sexes body weight was reduced at both 10 and 75 mg/kg bw/day, these decreases were within historical control data and did not show a dose-response relationship, and were therefore considered not to be related to treatment. There was no effect of treatment on the number of corpora lutea, implant sites, early or late resorptions, number of live or dead fetuses, or percent pre- or post-implantation losses per dam. There was also no effect at any group on the percentage of male fetuses.

There was no effect of treatment on the incidence of external malformations. Two fetuses were observed to have malformations, one at 75 mg/kg bw/day with umbilical hernia and one in the control group with gastroschisis median, spina bifida, hindlimb hyperflexion, and acaudate. As these findings showed no dose-relationship, they were considered not to be treatment-related. The incidence of runt fetuses was biologically significantly increased at 250 mg/kg bw/day when compared to control animals. There were no findings at visceral examination which indicated an effect of treatment in any group. There were no treatment-related skeletal malformations observed. A number of treatment-related skeletal anomalies and variants were recorded.

Conclusions

Maternal findings included decreased food consumption and body weight gain at 250 mg/kg bw/day. The maternal NOAEL was therefore 75 mg/kg bw/day. In fetuses, the incidence of runt fetuses was clearly increased at 250 mg/kg bw/day. The incidence of unilateral or bilateral 13th ribs was increased in all dose groups. However, the incidence of this same finding in the dams ranged from 77-96% in the four study groups with no effect on reproductive capability or normal life, thus observation of more than 12 ribs on each side of the rib cage was considered not to be an adverse finding. The fetal NOAEL, based on altered ossification patterns and on minor skeletal variants and anomalies, was 10 mg/kg bw/day.

Justification for classification or non-classification

The available data on toxicity to reproduction do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.