Registration Dossier

Administrative data

Description of key information

subacute toxicity:

OECD 410, dermal, rat, 28-days: NOAEL general toxicity 10 mg/kg bw/day

subchronic toxicity:

OECD 408, oral, rat, 90-days: NOAEL 30 ppm (equivalent to 1.96 mg/kg bw/day in males and 2.32 mg/kg bw/day in females)

chronic toxicity:

OECD 453, oral, rat, 6 months: NOAEL 25 ppm (eqivalent to 1.4 mg/kg bw/day in males and 1.8 mg/kg bw/day in females)

OECD 453, oral, rat, 12 months: NOAEL 25 ppm (equivalent to 1.1 mg/kg bw/day in males and 1.5 mg/kg bw/day in females)

OECD 453, oral rat, 24 months: NOAEL 25 ppm (equivalent to 1.0 mg/kg bw/day in males and 1.4 mg/kg bw/day in females)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
System:
other: exocrine pancreas, kidney
Organ:
kidney
pancreas

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
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 VIII - IX, 8.6, of Regulation (EC) No 1907/2006.
System:
other: gastrointestinal, hepatobiliary, endocrine
Organ:
liver
pancreas
pituitary gland

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
15 mg/cm²
Study duration:
subacute
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 VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Additional information

Reliable studies on the repeated dose toxicity of (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone are available. The data comprises a dermal short-term repeated dose toxicity study as well as oral short-term, subchronic and chronic repeated dose toxicity studies. All studies were performed according to GLP. Table 1 summarizes the repeated-dose toxicity studies performed with the test substance.

Table 1: Repeated-dose toxicity studies performed with (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone

 

Species and study type

NOAEL

LOAEL

Findings at LOAEL

Study ID

Mouse

90-day dietary
100, 750, 1500, 3000 ppm

3000 ppm

(M: 617 mg/kg bw/day, F: 500 mg/kg bw/day,)

> 3000 ppm

(M: > 500 mg/kg bw/day, F: > 617 mg/kg bw/day)

No relevant effects observed

M-103284-01-4

Rat

28-day dermal
10, 100, 1000 mg/kg bw/day

10 mg/kg bw/day

100 mg/kg bw/day

Increased liver weight, slight histopathological effects in pancreas and thyroid

M-263216-01-2

90-day dietary
2, 30, 1000, 7000 ppm

30 ppm

(M: 1.96 mg/kg bw/day, F: 2.32 mg/kg bw/day)

1000 ppm

(M: 66 mg/kg bw/day, F: 77 mg/kg bw/day)

Increased liver and kidney weights, histopathological effects in the kidney, cholesterol, triglycerides

M-102924-01-2

6-month rat dietary
25, 250, 1000, 2500 ppm

25 ppm
(M: 1.4 mg/kg bw/day, F: 1.8 mg/kg bw/day)

250 ppm
(M: 14 mg/kg bw/day, F: 19 mg/kg bw/day)

Decreased body weights, increased cholesterol, reduced urinary pH, increased liver, kidney and thyroid weights, histopathological effects in cornea, liver and thyroid

M-267037-02-1

12-month rat dietary
25, 250, 1000, 2500 ppm

25 ppm
(M: 1. mg/kg bw/day, F: 1.5 mg/kg bw/day)

250 ppm
(M: 11 mg/kg bw/day, F: 15 mg/kg bw/day)

Decreased body weights, increased cholesterol, reduced urinary pH, increased liver and kidney weights, histopathological effects in cornea, liver, kidney and thyroid

M-267037-02-1

24-month rat dietary

25, 250, 1000, 2500 ppm

25 ppm
(M: 1.0 mg/kg bw/day, F: 1.4 mg/kg bw/day)

250 ppm
(M: 10 mg/kg bw/day, F: 14 mg/kg bw/day)

Decreased body weights, increased cholesterol, reduced urinary pH, increased liver and kidney weights, histopathological effects in cornea, kidney and thyroid

M-267037-02-1

Dog

90-day dietary

100, 500, 1000 ppm

1000 ppm
(M: 40 mg/kg bw/day, F: 33 mg/kgbw/day)

> 1000 ppm

(M: > 40 mg/kg bw/day, F: > 33 mg/kg bw/day)

No relevant effects

observed

M-257679-01-1

12-month dietary

250, 1000, 3000 ppm

1000 ppm

(M: 34 mg/kg bw/day, F: 33 mg/kg bw/day)

3000 ppm

(M: 101 mg/kg bw/day, F: 93 mg/kg bw/day)

 

M: increased liver, kidney weights, increased hepatocytomegaly

F: increased liver, thyroid weights, increased hepatocytomegaly

M-267321-01-1

In studies with (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone in rats that required repeated dosing, corneal opacities were noted. This applies also to the rat studies 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 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 rat repeated dose studies 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 / carcinogenicity 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.


Oral

90-day subchronic toxicity feeding study in the mouse

In this study, which was conducted according to OECD 408, the test substance was incorporated into rodent diet and administered to 10 male and 10 female C57BL/6J mice per dose group at concentrations of 0, 100, 750, 1500, and 3000 ppm for 90 days (M-103284-01-4). These concentrations provided doses of 0, 16.5, 124, 259, and 500 mg/kg bw/day for males and 0, 19.7, 152, 326, and 617 mg/kg bw/day for females. Observations and examinations included clinical signs, body weights, and food consumption, ophthalmological examinations, clinical chemistry, urinalysis, gross necropsy, selected organ weights and histopathological examination.

No treatment-related mortalities occurred during the study and no treatment-related clinical signs were noted. There were no effects on body weight, body weight gain or food consumption. There were no treatment-related changes in any clinical chemistry parameters. Urine pH was statistically significantly increased at 3000 ppm in females, but was not analyzed statistically in males due to the small number of values obtained. There were no treatment-related effects on organ weights, gross necropsy or histopathological findings.

Conclusions

The only treatment-related finding was an increase in urinary pH in females at 3000 ppm. The NOAEL for the study is therefore considered to be 3000 ppm (500 mg/kg bw/day in males and 617 mg/kg bw/day in females).

 

90-day subchronic toxicity feeding study in the rat

In this study that was performed according to OECD 408 (M-102924-01-2), the test substance was mixed into powdered rodent diet at dietary concentrations of 0, 2, 30, 1000, 7000, or 12000 ppm, to provide doses of 0.13, 1.96, 66, 454, and 830 mg/kg bw/day for males and 0.15, 2.32, 77, 537, and 956 mg/kg bw/day in females. Observations and examinations included clinical signs, detailed clinical examinations, neurological (reflex) examinations, body weights, and food consumption, ophthalmological examinations, clinical chemistry and hematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

There were no mortalities in the groups receiving 0, 2, 30, or 1000 ppm. At 7000 ppm, one male was found dead on day 8, one male was sacrificed for humane reasons on day 70, and one male was killed for humane reasons on day 83. At 12000 ppm, one female was sacrificed on day 13 for humane reasons. Four males in this treatment group were found dead (days 15, 41, 45, and 72), and two males were killed for humane reasons (days 43 and 64), It was decided to sacrifice the surviving males of this group on day 72. No clinical signs were noted at either 2 or 30 ppm. At 1000 ppm and above, the significant clinical findings were yellow-colored urine and white areas on the cornea. There were no effects noted at examination of reflexes in week 12. Body weight was biologically decreased in both males and females at higher doses, with both 7000 ppm and 12000 ppm decreasing body weight gain by more than 10% in females. There were occasional slight decreases in food consumption by both males and females at 7000 and 12000 ppm, but there was no alteration in food consumption at concentrations of 1000 ppm or below. Neovascularization of the cornea and characteristic "snowflake" corneal opacities were noted at 1000 and 12000 ppm in males, and 1000 ppm, 7000, and 12000 ppm in females. There were no biologically significant effects on hematological parameters at any dose in either males or females. In males, bilirubin, aspartate and alanine aminotransferases, urea, and creatinine were biologically and or statistically significantly increased at the dietary concentration of 7000 ppm, while cholesterol and triglycerides were increased at 1000 and 7000 ppm. Alkaline phosphatase was decreased at 1000 ppm and above when compared to controls. Very few of these parameters showed any response in females. At urinalysis, ketone levels were increased from 1000 ppm in both males and females. This is likely due to detection of the triketone structure of the test substance itself, as the vast majority of the parent molecule is excreted in the urine unchanged. There was an increased incidence of occult blood, erythrocytes, leukocytes, and epithelial cells in the urine in both males and females at 7000 ppm and in females at 12000 ppm (males in the 12000 ppm group did not survive until the end of the study and urine was therefore not collected). Mean absolute and relative liver and kidney weights were biologically and statistically significantly increased in both males and females at 1000 ppm and above. In male rats either found dead or sacrificed prior to the end of the study (3 at 7000 ppm and 10 at 12000 ppm), the principal cause of death was considered to be related to calculi in the urinary tract. Clearly treatment-related macroscopic findings seen in these animals included gritty content in and dilation of the renal pelvis, obviously large kidneys, pale or mottled color of the kidneys, and foci on the kidneys, and red or gritty content in the urinary bladder, distension of the urinary bladder, and red foci in the bladder, and obviously large liver. Other findings which are less clearly related to treatment included dilation, dark content, and black foci of the stomach, soiled fur, and dark content in the intestines. In animals which survived to the end of the study, treatment-related macroscopic findings were seen at 1000 ppm and above. In both males and females from 1000 ppm, these findings included corneal opacities, abnormal shape of the kidneys, mottled kidneys, dilation of and gritty content in the renal pelvis, gritty content and distension of the urinary bladder, and gritty content of the ureters. A few females at 7000 and 12000 ppm were noted with obviously small kidneys, and one female at 12000 ppm showed gritty content in the urethra. A few males had obviously large liver at 1000 ppm, with prominent lobulation at 1000 and 7000 ppm. One male had obviously large thyroid at 1000 ppm. Microscopic pathology: The dose of 12000 ppm was above the Maximum Tolerated Dose in the rat and therefore histopathological examinations were not conducted at this dose. Additionally, as 30 ppm was observed to be free of treatment-related effects, histopathological examination was not conducted at 2 ppm. Treatment-related changes observed in final-sacrifice animals were within the urinary tract and the liver in both sexes, and the eyes and thyroid gland in males.

Conclusions

The primary effects of the test substance after subchronic dietary administration in the rat were limited to the eyes, liver, and urinary tract in both sexes and thyroid gland in males. Characteristic corneal "snowflake" lesions were observed in both males and females at doses of 1000 ppm and above (not relevant to humans). Increased liver weights were correlated with an increased incidence of centrilobular hypertrophy, with the greatest increase in incidence observed in males. Increased cholesterol and triglyceride concentrations were also observed in males at 1000 and 7000 ppm. Urinary tract stones (found to be primarily composed of the test substance, which has been demonstrated to be excreted in the urine at high concentrations after oral dosing) were observed in both males and females and were related to the histopathological findings including urothelial hyperplasia. In the thyroid, males at 1000 ppm and above showed increased incidences of follicular cell hypertrophy / hyperplasia and diffuse loss of colloid (not relevant to human).

Based on these findings, the NOAEL for dietary administration of the test substance in the male and female rat was 30 ppm (1.96 mg/kg bw/day in males, 2.32 mg/kg bw/day in females).

 

6-month chronic toxicity feeding study in the rat

In the 6-month toxicity phase of this combined chronic and carcinogenicity study (M-267037-02-1), that was performed according to OECD 453, the test substance was administered at dietary concentrations of 0, 25, 250, 1000, and 2500 ppm to groups of 10 male and 10 female Wistar rats for 6 months. These dietary concentrations provided doses of 1.4, 14, 58 and 143 mg/kg bw/day for males and 1.8, 19, 77 and 191 mg/kg bw/day for females. Observations and examinations included clinical signs, detailed clinical examinations, body weights, and food consumption, ophthalmological examinations, clinical chemistry and hematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

A treatment-related increase in mortality was observed in males at 2500 ppm (4/75 deceased animals versus 1/75 in the control), there was no treatment-related effect on mortality in females at any dose level. Treatment-related clinical signs of toxicity included white area on the eye and soiled fur in one or more areas. An increased incidence of these findings was observed at 250 ppm and above in both males and females. Body weight and body weight gain were reduced in males and females at the higher doses, with a greater effect observed in males than in females. There was no effect on food consumption at any dose in either males or females.

There was no treatment-related effect on hematology. Plasma cholesterol was biologically and statistically significantly increased at 250 ppm and above. This effect on plasma cholesterol was considered to be the only treatment-related clinical chemistry finding. Although plasma cholesterol was statistically significantly increased in males at 25 ppm, this value was within historical control data and cholesterol concentrations in this dose group returned to control levels for males of the other, longer two study phases, this increase was considered not to be treatment-related.

During urinalysis, higher ketone concentrations were noted in both males and females at 1000 ppm and above. These were due to the triketone form of the molecule, which is largely excreted unchanged in the urine. Urine pH was decreased at 250 ppm and above in males at all time points, while in females at 250 ppm and above urine pH was decreased only at the 3-month time point. Urinary protein was increased in males at all doses.

Liver and kidney weight was statistically significantly increased in males at 250 ppm and above, and thyroid weights was slightly increased at 250 ppm and above. There were no biologically significant effects on organ weights in females at any group or time point.

At necropsy, treatment-related findings were observed in the eyes, liver, and kidney. Eye opacities seen in both males and females were related to increased tyrosinemia and are considered not to be relevant for human risk assessment. Enlarged liver was observed in males at all doses, with prominent lobulation at 25, 250, and 1000 ppm.

Non-neoplastic microscopic findings: Treatment-related findings were observed in the eyes, liver, pancreas, thyroid gland, and kidneys. In the eyes, the incidence of corneal inflammation was increased in males at dietary concentrations of 250 ppm and above, and in females at 1000 and 2500 ppm. Regenerative hyperplasia of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm only. Neovascularization of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. Centrilobular hepatocellular hypertrophy was increased in males at all doses and in females from 250 ppm. Centrilobular hepatocellular vacuolation increased in incidence in males from 250 ppm, but was not observed in females. In the pancreas, an increased incidence of diffuse acinar degeneration / atrophy was reported in both males and females at 2500 ppm. In the thyroid, the incidence of altered colloid was increased in males in all treatment groups. The incidence of increased follicular diameter was increased in males from 250 ppm, but this finding was not observed in females. Pigment deposition in the follicular cells was increased in males from 250 ppm. The incidence of chronic progressive nephropathy was slightly increased in males at 2500 ppm.

Conclusions

Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. Similarly, the findings of thyroid colloid alteration and pigment deposition were considered to be specific to the rat and not indicative of an adverse finding, but to be normal findings in ageing rats. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL in this study was therefore considered to be 25 ppm (1.4 mg/kg bw/day in males and 1.8 mg/kg bw/day in females), while the LOAEL was 250 ppm (14 mg/kg bw/day in males and 19 mg/kg bw/day in females).

12-month chronic toxicity feeding study in the rat

In the 12-month toxicity phase of this combined chronic and carcinogenicity study (M-267037-02-1), that was performed according to OECD 453, the test substance was administered at dietary concentrations of 0, 25, 250, 1000, and 2500 ppm to groups of 10 male and 10 female Wistar rats for 12 months. These dietary concentrations provided doses of 1.1, 11, 45 and 114 mg/kg bw/day for males and 1.5, 15, 63 and 155 mg/kg bw/day for females. Observations and examinations included clinical signs, detailed clinical examinations, body weights, and food consumption, ophthalmological examinations, clinical chemistry and hematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

 A treatment-related increase in mortality was observed in males at 2500 ppm (8/65 deceased animals versus 2/65 in the control). There was no treatment-related effect on mortality in other male treatment groups or in females at any dose level. Treatment-related clinical signs of toxicity included white area on the eye and soiled fur in one or more areas. An increased incidence of these findings was observed at 250 ppm and above in both males and females. Body weight and body weight gain were reduced in males and females at the higher doses, with a greater effect observed in males than in females. There was no effect on food consumption at any dose in either males or females.

There was no treatment-related effect on hematology. Plasma cholesterol was biologically and statistically significantly increased at 250 ppm and above. This effect on plasma cholesterol was considered to be the only treatment-related clinical chemistry finding. During urinalysis, higher ketone concentrations were noted in both males and females at 1000 ppm and 2500 ppm. These were due to the triketone form of the test substance, which is largely excreted unchanged in the urine. Urine pH was decreased at 250 ppm and above in males at all time points, while in females at 250 ppm and above urine pH was decreased only at the 3-month time point. Urinary protein was increased in males at all doses from month 7 onwards.

Liver and kidney weight was statistically significantly increased in males at 250 ppm and above. There were no effects on organ weights in females. At necropsy, treatment-related findings were observed in the eyes, liver, and kidney. Eye opacities seen in both males and females were related to increased tyrosinemia and are considered not to be relevant for human risk assessment. Enlarged liver was observed in males at 250 ppm and above.

Non-neoplastic microscopic findings: Treatment-related findings were observed in the eyes, liver, pancreas, thyroid gland, and kidneys. Findings observed in other organs were considered to be of little to no toxicological relevance or not to be treatment-related. In the eyes, the incidence of corneal inflammation was increased in males at dietary concentrations of 250 ppm and above, and in females at 1000 and 2500 ppm. Regenerative hyperplasia of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. Neovascularization of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. Centrilobular hepatocellular hypertrophy was increased in males only at 250 ppm and above. Centrilobular hepatocellular vacuolation increased in incidence in males from 250 ppm, but was not observed in females.

In the pancreas, diffuse acinar degeneration / atrophy was reported in males at 1000 and 2500 ppm, and in females at 2500 ppm. In the thyroid, the incidence of altered colloid and increased follicular diameter was increased in males from 250 ppm. Pigment deposition in the follicular cells was increased in males from 250 ppm. The incidence of focal follicular cell hyperplasia was slightly increased in males at 2500 ppm. Diffuse follicular cell hypertrophy was noted in a few males and females from 250 ppm.

The incidence of chronic progressive nephropathy was slightly increased in males from 250 ppm and in females at 250 and 1000 ppm (one animal each). Neoplastic microscopic findings: A small number of tumors was noted in animals from treated and control groups with no evidence of an effect of treatment.

Conclusions

Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL in this study was therefore considered to be 25 ppm (1.1 mg/kg bw/day in males and 1.5 mg/kg bw/day in females), while the LOAEL was 250 ppm (11 mg/kg bw/day in males and 15 mg/kg bw/day in females).

24 -month chronic toxicity feeding study in the rat

In this study conducted according to OECD 453, (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone was incorporated into rodent diet and administered at concentrations of 0, 25, 250, 1000, and 2500 ppm to groups of 75 male and female Wistar rats (M-267037-01-2). In addition to the 24 -month phase, the study also included a 6-month and 1-year repeated-dose toxicity phase.

The administered concentrations provided final doses of 0, 1.0, 10, 41, and 105 mg/kg bw/day for males and 0, 1.4, 14, 57, and 141 mg/kg bw/day for females. Body weight and food consumption were measured weekly for the first 13 weeks of the study, then every 4 weeks through the remainder of the study until necropsy. Ophthalmological examinations were conducted on all animals during acclimatization and at months 3, 6, 12, 18, and 24 of treatment. Blood for hematological and clinical chemical examination was collected after 6, 12, and 24 months from overnight-fasted rats. Urine was collected overnight at 3, 6, 12, 18, and 24 months from animals fasted overnight of food and water.

A treatment-related increase in mortality was observed in males at 2500 ppm. There was no treatment-related effect on mortality in other male treatment groups or in females at any dose level. Treatment-related clinical signs of toxicity included white area on the eye and soiled fur in one or more areas. An increased incidence of these findings was observed at 250, 1000, and 2500 ppm in both males and females. Body weight and body weight gain were reduced in males and females at the higher doses, with a greater effect observed in males than in females. There was no effect on food consumption at any dose in either males or females.

Treatment-related findings (corneal opacity, neovascularization of the cornea, oedema of the cornea, and "snow flake" corneal opacities) were seen during ophthalmoscopy in both males and females. At 6 and 12 months, increased incidence of these findings was seen only at 250, 1000, and 2500 ppm. At 24 months, these findings were increased compared to controls at all doses in males. However, at 25 ppm there was only a slight increase in corneal opacity, neovascularization of the cornea, and oedema of the cornea in males in neovascularization of the cornea in females. The findings are related to tyrosinemia resulting from inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPDase), a key enzyme in the tyrosine catabolic pathway. As humans are capable of metabolizing and excreting excess tyrosine while rats are much less capable of this metabolism, the corneal effects of HPPDase inhibitors are considered to be relevant for rats but not for man (for a detailed justification see endpoint summary for repeated dose toxicity).

There was no treatment-related effect on hematology at any dose or time point in either male or female rats. Plasma cholesterol was biologically and statistically significantly increased at 250 ppm and above at months 7 and 12, and was biologically and/or statistically significantly increased at 1000 and 2500 ppm at 18 and 24 months. This effect on plasma cholesterol was considered to be the only treatment-related clinical chemistry finding. Although plasma cholesterol was statistically significantly increased in males at 25 ppm at the 7-month time point, this value was within historical control data and cholesterol concentrations in this dose group returned to control levels by the 12- month time point; therefore, this increase was considered not to be treatment-related.

During urinalysis, higher ketone concentrations were noted in both males and females at 1000 ppm and 2500 ppm at all collection periods, and in males at 250 ppm at months 19 and 24 only. These were due to the triketone structure of the molecule, which is largely excreted unchanged in the urine. Urine pH was decreased at 250 ppm and above in males at all time points, while in females at 250 ppm and above urine pH was decreased only at the 3-month time point. Urinary protein was increased in males at all doses from month 7 onwards.

Liver and kidney weight was statistically significantly increased in males only at the higher doses in this study. There were no biologically significant effects on organ weights in females at any group. At necropsy, treatment-related findings were observed in the eyes, liver, and kidney. Eye opacities seen in both males and females were related to increased tyrosinemia and are considered not to be relevant for human risk assessment. An increased incidence of pale kidneys or irregular surface of the kidney was noted in males at 1000 and 2500 ppm.

 

Non-neoplastic microscopic findings: Treatment-related findings were observed in the eyes, liver, pancreas, thyroid gland, and kidneys. Findings observed in other organs were considered to be of little to no toxicological relevance or not to be treatment-related.

In the eyes, the incidence of corneal inflammation was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm. At 24 months, there was a very slight increase in corneal inflammation in males at 25 ppm and in females at 250 ppm. Regenerative hyperplasia of the cornea was increased in males at 250 ppm and above, and in females at 1000 and 2500 ppm, only. Neovascularization of the cornea was increased in males 250 ppm and above, and in females at 250 ppm and above. There was an increase in males in the incidence of mucous metaplasia of the cornea at 250 ppm and above. Corneal atrophy was increased in males at 250 ppm and above and in females at 1000 and 2500 ppm, and peripheral retinal atrophy was increased in both males and females at 250 ppm and above.

Centrilobular hepatocellular hypertrophy was increased in males from 250 ppm. In females centrilobular hepatocellular hypertrophy was only reported in one animal at 2500 ppm. In the pancreas, diffuse acinar degeneration / atrophy was reported in both males and females; the incidence of diffuse acinar degeneration / atrophy was increased in males and females at 1000 and 2500 ppm. The incidence of focal acinar degeneration / atrophy was increased in females at 2500 ppm.

In the thyroid, the incidence of altered colloid was increased in males and females from 250 ppm and above. Pigment deposition in the follicular cells was increased in both males and females in all dietary groups. The incidence of focal follicular cell hyperplasia was slightly increased in males from 250 ppm. In females, this finding was only at a very low incidence. No clear relationship of follicular cell hyperplasia to dose was evident. Diffuse follicular cell hypertrophy was noted in a few males and females from 250 ppm, again in the absence of a clear dose-relationship. The observed thyroid findings are considered a rat-specific phenomenon of HPPDase inhibitors without relevance for humans (for a detailed justification see endpoint summary for repeated dose toxicity).

There was no substantive dose-related increase in the incidence of chronic progressive nephropathy in males at 24 months, and the incidence in control animals at 24 months of 80% indicates that this is a very common finding in the aging male rat. Chronic progressive nephropathy was observed in females, with no dose relationship. Hyperplasia of the collecting ducts was increased in males at 1000 and 2500 ppm.

 

Conclusions

Based on increased mortality at 2500 ppm in males, this dose is considered to have exceeded the Maximum Tolerated Dose. Treatment-related findings were seen in the eyes, liver, kidney, thyroid, and pancreas. Findings in the eye were related to the biochemical mechanism of the test substance as an HPPDase inhibitor leading to increased tyrosinemia in rats, and were considered not to be relevant for humans. The observations in the liver, including centrilobular hepatocellular hypertrophy, were evaluated as being adaptive responses to treatment rather than adverse effects. The finding of chronic progressive nephropathy is considered to be a rodent-specific finding not relevant to man. Similarly, the findings of colloid alteration and pigment deposition were considered to be specific to the rat and not indicative of an adverse finding, but to be normal findings in ageing rats. The increased incidence of acinar degeneration / atrophy in the pancreas was only observed at 1000 and 2500 ppm, and may indicate a tyrosine-linked effect as well, as these findings were not observed in the mouse or dog, which are not as sensitive as the rat to the effects of tyrosinemia.

The NOAEL for general toxicity in this study was therefore considered to be 25 ppm (1.0 mg/kg bw/day in males and 1.4 mg/kg bw/day in females), while the LOAEL was 250 ppm (10 mg/kg bw/day in males and 14 mg/kg bw/day in females).

 

90-day subchronic toxicity feeding study in the Beagle dog

In this study, that was conducted according to OECD 452, the test substance was administered to groups of 4 male and 4 female beagle dogs by dietary admix at doses of 0, 100, 500 and 1000 ppm (M- 267321-01-1). These concentrations provided doses of 0, 3, 17, and 40 mg/kg bw/day for males and 0, 3, 17, and 33 mg/kg bw/day for females. Observations and examinations included clinical signs, body weights, and food consumption, ophthalmological examinations, clinical chemistry and hematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

All animals survived until scheduled necropsy and there were no treatment-related clinical signs in either males or females. Body weight and food consumption were unaffected. There were no ophthalmological findings at any dose level which were ascribed to treatment. There were no treatment-related hematological, clinical chemistry or urinalysis findings at any dose level. Gross necropsy finding, organ weights and histopathology revealed no treatment-related findings in either males or females at any dose.

Conclusions

In the absence of any findings, the NOAEL for this study was 1000 ppm (40 mg/kg bw/day in males and 33 mg/kg bw/day in females).

 

12-month chronic toxicity feeding study in the Beagle dog

In this study, which was performed according to OECD 452, the test substance was incorporated into diet and administered to groups of 4 male and 4 female beagle dogs at doses of 0, 250, 1000, and 3000 ppm for one year (M-267321-01-1). These concentrations provided doses of 0, 7, 34, and 101 mg/kg bw/day for males and 0, 9, 33, and 93 mg/kg bw/day for females. Observations and examinations included clinical signs, body weights, and food consumption, ophthalmological examinations, clinical chemistry and hematology, urinalysis, gross necropsy, selected organ weights and histopathological examination.

All animals survived until scheduled necropsy and there were no treatment-related clinical signs in either males or females. There were no biologically relevant effects on body weights and food consumption. There were no abnormalities noted at ophthalmological examination. There were no treatment-related hematological, clinical chemistry or urinalysis findings. Treatment-related changes in organ weights were noted for liver, thyroid, and kidney at 3000 ppm only, specifically increased absolute and relative liver weight in males and females, increased absolute and relative kidney weight in males, and increased absolute and relative thyroid weight in females. There were no observations at gross necropsy which were considered to be related to treatment. The only treatment-related histopathology findings were seen in the liver in male and female dogs at 3000 ppm, in which the incidence of centrilobular and/or midzonal hepatocytomegaly was increased (2 in males, 3 in females) compared to controls (0 in both males and females).

Conclusions

The toxicity of the test substance by dietary administration in the dog was limited. In this one-year study, administration of 3000 ppm caused increases in absolute and relative liver weights in males and females, absolute and relative kidney weights in males, and absolute and relative thyroid weights in females. The treatment-related histopathological lesions were limited to increased incidence of hepatocytomegaly in both males and females at 3000 ppm. The NOAEL for this study was 1000 ppm (34 mg/kg bw/day for males and 33 mg/kg bw/day for females).

 

Dermal:

In a 28-day subacute dermal toxicity study conducted according to OECD 410 the test substance was administered to rats (M-263216-01-2). The test substance was moistened with tap water and applied under a semiocclusive bandage to the shaved skin of 10 male and 10 female Wistar rats per treatment group, at dose levels of 0, 10, 100, and 1000 mg/kg bw/day. The substance was applied five days per week for the first three weeks of the study, and seven days per week for the final week, so that all animals were dosed daily for at least the last 8 days of the study. On each treatment day, the application period was 6 hours, after which remaining test substance was washed off with water. Observations and examinations included clinical signs, ophthalmological examinations in all high-dose and control animals, body weights, food and water consumption, evaluation eventual skin reactions, hematology, clinical chemistry, gross necropsy, selected organ weights and histopathology.

There were no mortalities or clinical signs, nor were treatment-related effects observed at ophthalmological investigation of either male or female rats at 1000 mg/kg bw/day. There were no treatment-related effects on body weights, food or water consumption, hematological and clinical chemistry parameters. Absolute and relative liver weight was statistically significantly increased in both males and females at 1000 mg/kg bw/day. Absolute liver weight was statistically significantly increased in females at 10 mg/kg bw/day, but as there was no dose-relationship and as body weight was also increased at this dose, this was considered not to be treatment-related but to be due to increased body weight. There were no observations at necropsy which were treatment-related.

There was no evidence of any reaction at the application sites to dermal application. Focal degeneration of the pancreas was observed in both males and females at 100 and 1000 mg/kg bw/day. Thyroid follicular cell hypertrophy was seen in both males and females in both control and treated animals. Alteration of thyroid colloid was seen at an increased incidence and severity in 100 and 1000 mg/kg bw/day in males. Hepatocellular hypertrophy and hypertrophy of the pars distalis of pituitary were observed in males at 1000 mg/kg bw/day. These findings in the pancreas, thyroid, pituitary and liver are considered to be treatment-related.

 

Conclusions

There were few effects of repeated dermal application of the test substance to either male or female rats. There were no local effects on either skinfold thickness or on signs of irritation. The local NOAEL was determined to be 1000 mg/kg bw/day (equivalent to 15 mg/m2 skin), while the systemic NOAEL was 10 mg/kg bw/day, based on histopathological effects observed at 100 and 1000 mg/kg bw/day in both males and females.

 

Justification for classification or non-classification

Based on the effects observed in the oral and dermal repeated dose toxicity study at 1000 ppm (M: 66 mg/kg bw/day; F: 77 mg/kg bw/day) and 100 mg/kg bw/day, respectively, the substance is classified “STOT RE 2, H373”.