5-Brom-2-[(3,4,5-trifluorphenoxy)-difluormethyl]-1,3-difluorbenzene

Endpoint summary

Description of key information

Repeated dose toxicity (OECD 407, RL1): rat (m/f): NOAEL = 300 mg/kg bw/d (highest dose tested).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Dec 2015 - 07 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on species / strain selection:

For the testing of chemicals in 28-day oral repeat-dose toxicity studies in rodents, the preferred species is the rat according to the guidelines mentioned above.
The Wistar WI (Han) rat was chosen since it is widely accepted by Authorities as an appropriate experimental model, with documented susceptibility to a wide range of toxic substances.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 8-9 weeks (males/females)
- Weight at study initiation: males: 216 - 254 g; females: 155 - 177 g
- Fasting period before study: Before blood sampling, the animals were kept in metabolism cages for the collection of urine for approximately 18 hours without food.
- Housing: gang-housed (2 or 3 animals/sex) in type IV Makrolon® cages. During the short time period between functional observations and motor activity, the animals were kept individually in type III Makrolon® cages.
- Diet (e.g. ad libitum): Ssniff V1534 diet (batch 1324730) ad libitum
- Water (e.g. ad libitum): tap water (ad libitum); Drinking water was available from Makrolon® drinking bottles freshly filled twice a week.
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY: The diet is checked periodically by an independent and approved laboratory, according to the specifications of the manufacturer. Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides, and antibiotics. The drinking water is periodically analyzed according to the German regulations for human drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 40 - 70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally by gavage (flexible tube), once daily, for 28 consecutive days. The control animals were treated at the same frequency with the vehicle.
The volume of administration was 5 mL/kg body weight. The volume of administration per animal was calculated by means of the LIM-System (Provantis®).

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared freshly each day of use.
The preparations of the test item formulations were stored under normal room conditions and constant magnetic stirring. Exposure to light was kept to a minimum.
The preparation of the test item formulations was performed by the Central Dispensary of the test facility.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

A concomitant homogeneity/stability study showed homogeneous distribution and chemical stability of the test item in the vehicle aqueous 0.5% hydroxypropyl methylcellulose (Methocel® K4M Premium) at concentrations of 2, 20 and 60 mg/mL stored at room temperature for 1 day.
It was decided to omit formulation analysis and prepare the test item formulations freshly each day.

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

once daily

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

corresponds to 6 mg/mL

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

corresponds to 20 mg/mL

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

corresponds to 60 mg/mL

No. of animals per sex per dose:

Main group:
The control and high dose groups: 10 male and 10 female rats each.
The low and mid dose groups: 5 male and 5 female rats each.
Recovery group:
5 male and 5 female rats each in the control group and high dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The test item was tested for acute toxicity in Wistar rats (HsdCpb:WU) after single oral dose of 2000 mg/kg bw. No mortality or signs of toxicity were seen in the rats (3 males, 3 females). Therefore, the LD50 was considered to be higher than 2000 mg/kg bw after single oral administration.
The test item was tested in a 28-day dose range finding study in Wistar rats (Crl:WI (Han)) at 300 and 1000 mg/kg bw/d. At 1000 mg/kg bw/d all rats (3 males, 3 females) were prematurely sacrificed between day 6 and 7. However, a dose of 300 mg/kg bw/d was clinically well tolerated for 28 days and at necropsy the only macroscopic finding was an enlargement of the liver observed in all rats (3 males, 3 females).
Therefore, for the current study, 300 mg/kg bw/d was selected as the high dose with the aim of inducing mild to moderate systemic toxic effects. The dose level of 100 and 30 mg/kg bw/d were selected as the mid and low dose, respectively, to investigate dose-dependent effects and to define a NOAEL.

Positive control:

none

Observations and examinations performed and frequency:

Mortality
Mortality of each animal was checked daily. The parameter was recorded with the LIM-System.

Clinical Signs
The behavior and appearance of each animal were checked daily. The parameters were recorded with the LIM-System.

Body Weight
Body weight was recorded before treatment and thereafter weekly with the LIM-System. Body weight gain is a calculated parameter (difference to baseline value) that was determined for each animal. The parameter was calculated for different intervals, e.g. weekly or total, with the LIM-System.

Food and Water Consumption
Food Consumption
Food consumption was determined once a week by weighing the food per cage which had not been consumed. The parameter was recorded with the LIM-System.

Water Consumption
Water consumption was determined twice a week by measuring the water per cage which had not been consumed. The parameter was recorded with the LIM-System.

Detailed Clinical Observations
In addition to the daily observation of clinical signs detailed clinical observations were performed in all main and recovery group animals before the first dose (day -1) and in weeks 1, 2, 3 and 4. For the detailed clinical observations the animals were taken out of the home cage, palpated and then transferred in a separate type IV Makrolon® cage.

Motor Activity
In week 4, one hour after administration, the main group animals (5 males and 5 females per group; see table below) were removed from their home cages and their motor activity was recorded in special motor activity cages over 60 minutes at 5 minutes intervals (ActiMot-System by TSE Systems, Bad Homburg, Germany). The number of movements was evaluated by counting the number of interruptions of photo beams (7 beams on the x-axis, 4 beams on the y-axis, 7 beams on the z-axis). This parameter is called “counts” and given in numbers. The system calculated the “on-time” (minutes), “off-time” (minutes), the “total distance” the animal was moving (meter), “rearing” (number), and “rearing time” (minutes). The data was transferred into the LIM-System. Assignment of the rats to the individual measurements followed a randomization schedule. On each measuring day, measurements were conducted simultaneously in 10 rats.

Functional Observational Battery (FOB)
A functional observational battery (FOB), however, without descriptive parameters, was performed in the main group animals. The FOB was performed after the motor activity measurement. Animal numbers and micro transponder implants were not identified for the laboratory staff performing the functional observational battery. Therefore, the observers did not know to which treatment group the rats belong. The FOB parameters were recorded with the LIM-System.

Laboratory Tests
In weeks 5 and 7 approximately 2.5 mL blood was taken sublingually under inhalation anesthesia (isoflurane) from the rats as listed below. The blood samples were divided for hematological and clinico-chemical examinations. Before blood sampling the rats were kept on metabolism cages for approximately 18 hours without food for urine sampling.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Rats were designated for pathology examination after the end of the treatment (main kill) or recovery period. The animals were necropsied, examined for gross pathological alterations, the weights of selected organs were recorded); Selected organs: Heart, Liver, Kidneys, Spleen, Thymus, Testes, Prostate, Uterus, Ovaries, Adrenals, Thyroids with Parathyroids, Brain, Epididymes, Seminal vesicles

HISTOPATHOLOGY: Yes (Adrenal, Aorta, Bone (femur), Bone marrow (sternum, femur), Brain (cerebrum, cerebellum, brain stem), Esophagus, Eye, Heart, Instestine (large and small), Kidney, Knee joint, Larynx, Liver, Lung, Lymph nodes, Mammary gland, Mico transponder, Muscle (skeletal, thigh), Nasal turbinates, Nerve (optic and sciatic), Pancreas, Parathyroid, Peyer's Patches, Pituitary, Reproductive organs (ovary, oviduct, uterus, vagina, epididymis, prostate, seminal vesicle, testis), Salivary gland, Skin (inguinal), Spinal cord, Spleen, Stomach, Thymus, Thyroid, Tongue, Trachea, Ureter, Urinary bladder, Zymbal's gland

Statistics:

Body weight, body temperature, food and water consumption, organ weights, hematology, clinical chemistry, motor activity, functional observational battery (numerical parameters):
To compare the treatment groups with the control group, the following statistical procedures were applied separately for each sex and each measuring point. To take the number of dose groups into account all the test procedures used maintain a multiple significance level of alpha = 0.05.
Absolute body weight, body weight gain (differences to baseline values on day 1), body temperature, food and water consumption, organ weights (relative and absolute), clinical pathology parameters (hematology, clinical chemistry serum parameters, specific gravity and urine weight), and motor activity (total time period of 60 minutes), dose groups were compared with those of the control, using the multiple two-sided Dunnett-Test.
For all numerical parameters of the FOB, dose groups and the control groups were compared, using the non-parametric 2-sided Kruskal-Wallis test followed by the Wilcoxon-test rank sum test.
For all parameters (for urinalysis only specific gravity and urine weight) mean values, standard deviation (except for numerical FOB parameters), and number of animals (N) were calculated.
For each numerical parameter of the FOB also minimum, maximum, median, and the frequencies of scores were calculated.
Software: Body weight, body temperature, food and water consumption, organ weights, hematological parameters, clinico-chemical parameters (for urinalysis only specific gravity and urine weight), motor activity and the FOB parameters (except the frequencies of scores) will be evaluated within the LIM-System. The urinalysis examinations and detailed clinical observations were recorded with the LIM-System.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One 100 mg/kg bw/d female (No. 32) showed a skin swelling of the hindleg from day 8 to 10. This symptom occurred after the animal accidently trapped its hindleg in the cage lid. Therefore, this symptom, which recovered after a few days, is not considered treatment-related.
Four 300 mg/kg bw/d females showed hair loss. These findings were without a dose relationship and also observed in one male of the control group. Therefore, these findings are considered incidental.
Immediately after oral administration salivation was observed for less than 5 minutes on several occasions during the treatment period in four 300 mg/kg bw/d males. Short-term salivation immediately after oral administration indicates rather a bad taste of the test item preparation than a toxicological effect in rats.
One 300 mg/kg bw/d male showed a mutilated foreleg. This finding was already observed on day 1 before start of treatment. Therefore, these finding is not considered treatment-related.

On day 28 hair loss was observed in one control male (0 mg/kg bw/d; animal No. 43) and in three 300 mg/kg bw/d females (animal Nos. 36, 39, 40).
On day 21 hair loss was observed in two 300 mg/kg bw/d females (animal Nos. 36, 39).
Hair loss was also observed in one male of the control group und no dose relationship was seen. Therefore, these findings are considered incidental.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed, all animals survived until their scheduled sacrifice.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight gain (absolute and relative compared to control): Day 1-42
In 300 mg/kg bw/d males the mean body weight gain was slightly (11.9%) decreased compared to control groups at the end of the recovery period on day 42, however, without reaching statistical significance.
All other changes in mean body weight gain are not considered treatment-related since they are they are not statistically significant, do not differ from changes/variations in control animals and/or do not show any dose-dependency.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was slightly decreased in week 1 (day 1-7) in 300 mg/kg/d females, in week 3 (day 14-21) in 100 mg/kg bw/d males and in week 4 (day 21-28) in 100 mg/kg bw/d males. These effects are not considered treatment-related, because they were not dose dependent and did no show a correlation to effects on body weight/body weight gain.
During the recovery period no effects on food consumption were observed.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption was statistically significantly decreased in 30 mg/kg/d males from days 10-14, in 100 mg/kg bw/d males from days 7-14 and 300 mg/kg bw/d males from days 4-10. Water consumption was statistically significantly increased in 300 mg/kg bw/d males from days 24-31 and 35-42. In females, water consumption was statistically significantly decreased in the 300 mg/kg bw/d group from days 1-7, 24-28 and from days 35-38.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology and coagulation measurements showed no findings during the treatment and treatment-free recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry evaluation revealed a slight increase of chloride (up to 1.06-fold) and cholesterol (up to 2.57-fold) in both sexes at 100 and 300 mg/kg bw/d at the end of the treatment period (day 29). Additionally, total protein was slightly (1.07-fold) increased in 100 mg/kg bw/d males and in both sexes at 300 mg/kg bw/d. An increase of bile acids was observed in 300 mg/kg bw/d males only. At the end of the treatment-free recovery period (day 43) a partial recovery of effects was observed. However, at 300 mg/kg bw/d bile acids (1.62-fold) and total protein (1.04-fold) were still slightly increased in males and chloride (1.02-fold) and cholesterol (1.41-fold) in females.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis did not reveal any treatment-related effects.

Description (incidence and severity):

Autonomous Nervous System
No significant effects on lacrimation, salivation, pupil response, piloerection, defecation (number of fecal boluses, feces consistency), urination (number of urine pools, urine stain size), and palpebral closure were seen in any dose group. One 100 mg/kg bw/d male (animal No. 15) showed soft feces. This was a single event and no dose dependency was observed. Therefore, the finding is not considered treatment-related.
Neuromuscular System
No statistically significant effects on gait, hindlimb grip strength, hindlimb foot splay, righting reflex, mobility, or muscle tone were observed in all dose groups.
Forelimb grip strength was significantly decreased in 100 mg/kg bw/d males. Since no dose dependency was observed, this finding is not considered treatment-related.
Sensomotoric System
No statistically significant effects on approach response, click response, touch and tail pinch response were observed in all dose groups.
Central Nervous System
No statistically significant effects on ease of removal and handling, arousal, fur appearance, raising, raising behavior, or catalepsies could be observed in all dose groups.
Body Temperature
No significant changes of body temperature in treated animals versus control were observed on day 28.
Motor Activity
Motor activity in males and females evaluated as counts in 5-minute segments was not different in treatment groups compared to controls over time. All additional parameters of motor activity – off-time (minutes), on-time (minutes), rearing no., rearing time (minutes), total distance (meter) – measured in week 4 did not show treatment-related differences of treatment groups versus control.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The organ weights of the main kill animals revealed a dose-dependent increase of liver weights in both sexes. In 30 mg/kg/d animals, only a trend (without statistical significance) towards increased liver weights, 25% in males and 9% in females, was seen. The absolute liver weight increase in males was about 58% in 100 mg/kg bw/d animals and 91% in 300 mg/kg bw/d animals. In females an absolute liver weight increase of about 42% was observed at 100 mg/kg bw/d and 68% at 300 mg/kg bw/d. Thyroid weights were increased in males at 100 and 300 mg/kg bw/d. The absolute thyroid weight increase achieved approximately 31% at 100 mg/kg bw/d and 72% at 300 mg/kg bw/d.
The observed liver and thyroid weight increases were not completely reversible within the 2- week treatment-free recovery period. At 300 mg/kg bw/d the liver weights were still increased by approximately 27% in males and 23% in females and the thyroid weights by approximately 51% in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At gross pathology, the main kill animals (day 29) showed a dose-dependent increase of liver size in all dose groups. At recovery kill (day 43) an increased liver size was still detectable in the 300 mg/kg bw/d animals.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology of the main kill animals revealed treatment-related lesions in the liver, thyroids and kidneys at all dose levels with a dose-dependent increase in incidence and severity.
At 30 mg/kg bw/d a minimal to mild centrilobular hepatocellular hypertrophy occurred in 3/5 males and 2/5 females. Minimal to mild follicular hyperplasia/hypertrophy of the thyroid gland was seen in 3/5 males and 2/5 females. In the proximal tubules of the kidney 3/5 males showed a minimal eosinophilic droplet accumulation.
At 100 mg/kg bw/d, a moderate centrilobular hepatocellular hypertrophy was detected in all animals. In the thyroid gland, 4/5 males and all females showed a mild to moderate follicular hyperplasia/hypertrophy. A minimal condensation of colloid was seen in one male. In the kidney a mild to moderate accumulation of eosinophilic droplets within the proximal tubules was detected in 4/5 males.
At 300 mg/kg bw/d histopathology showed a marked centrilobular hepatocellular hypertrophy in all animals. One female exhibited mildly increased numbers of single cell necrosis in the centrilobular area. A mild to marked follicular hyperplasia/hypertrophy of the thyroid gland was detected in all animals. However, the severity of this finding was higher in males. A minimal to mild condensation of colloid was seen in all males whereas females were unaffected. In the kidney, a mild to moderate accumulation of eosinophilic droplets in the tubular epithelium was observed in males only.
Histopathology of 300 mg/kg bw/d recovery kill animals showed a minimal to moderate centrilobular hepatocellular hypertrophy in all animals and increased numbers of single cell necrosis in the centrilobular area in 3/5 males with a minimal to mild severity. A minimal to moderate follicular hyperplasia/ hypertrophy of the thyroid gland was detected in 4/5 males and 3/5 females. In the proximal tubules of the kidney a mild eosinophilic droplet accumulation was seen in all males.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Daily oral treatment with the test item at doses of 30, 100 and 300 mg/kg was tolerated over 4 weeks. In all dose groups histopathological investigations revealed centrilobular hepatocellular hypertrophy, follicular hyperplasia/ hypertrophy of the thyroid gland, condensation of colloid and accumulation of eosinophilic droplets in the tubular epithelium of the kidneys. All findings exhibited a clear dose-dependency with regard to incidence and severity and are not considered to represent adverse effects but rather reflect metabolic adaptive changes.
After the two week recovery period, the same findings were observed as in main kill animals, but with less pronounced severity. Therefore only a tendency towards reversibility could be shown.
Centrilobular liver cell hypertrophy is an adaptive metabolic response after exposure to a microsomal enzyme inducing xenobiotic. The follicular hypertrophy/hyperplasia in the thyroid gland is a consequence of increased metabolism of thyroid hormones in the liver. It is well established that the rat is especially prone to this thyroid activation. The cause for the eosinophilic droplet accumulation in the kidney of male rats could not be elucidated in the current study.
The alterations of cholesterol, total protein and total bile acid may be considered as an indicator of increased metabolism and are in line with the findings seen in histopathology (hepatocellular hypertrophy). The reason for the dose-dependent increase in chloride is unclear, because it was not accompanied by further pathological alterations (e.g. imbalance of other serum electrolytes) and recovered nearly completely.
The No Observed Adverse Effect Level (NOAEL) of the test item in Wistar (Han) rats was established at 300 mg/kg bw/d.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest dose tested

Key result

Critical effects observed:

no

Conclusions:

The test item was administered orally by gavage, once daily, 7 times a week, for 4 weeks to 3 groups of male and female Crl:WI (Han) rats at doses of 30, 100 or 300 mg/kg bw/d. A similarly constituted control group received the vehicle, 0.5% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium).
The control and high dose groups consisted of 10 male and 10 female rats each. The low and mid dose groups consisted of 5 male and 5 female rats each.
The No Observed Adverse Effect Level (NOAEL) in Wistar (Han) rats was established at 300 mg/kg bw/d.

Executive summary:

A subacute toxicity study in rats according to OECD TG 407 was conducted with the test item. 

The test item was administered orally by gavage, once daily, 7 times a week, for 4 weeks to 3 groups of male and female Crl:WI (Han) rats at doses of 30, 100 or 300 mg/kg.A

similarly constituted control group received the vehicle, 0.5% aqueous hydroxypropyl

methylcellulose (Methocel® K4M Premium).

The control and high dose groups consisted of 10 male and 10 female rats each. The low and mid dose groups consisted of 5 male and 5 female rats each. At the end of the treatment period 10 (5 males, 5 females) rats per group were scheduled for necropsy. The remaining rats of groups 1 and 4 were scheduled for a 2-week recovery period. The rats were gang-housed under conventional conditions.

At the end of the treatment or recovery periods, all animals were subjected to a detailed necropsy. Organ weights were recorded, and histopathological examinations performed on selected organs.

No mortality was observed, all animals survived until their scheduled sacrifice. The only treatment-related clinical sign in 300 mg/kg/d males was short-term salivation observed immediately after oral administration for less than 5 minutes on several occasions during the treatment period. Short-term salivation immediately after oral administration indicates rather a bad taste of the test item preparation than a toxicological effect in rats and is therefore not considered toxicologically relevant.

The detailed weekly clinical observations revealed not treatment-related findings. Mean body weight was slightly (5.1%) decreased compared to control groups at the end of the recovery period (day 42) in 300 mg/kg/d males without reaching statistical significance. Body weight gain decreased accordingly.

No effects on food consumption were observed during the treatment and treatment-free recovery period.

Water consumption was statistically significantly decreased in 30 mg/kg/d males from day 10 to 14 in 100 mg/kg/d males, from day 7 to 14, and in 300 mg/kg/d males from day 4 to 10. Water consumption was statistically significantly increased in 300 mg/kg/d males from day 24 to 31 and 35 to 42. In females, water consumption was statistically significantly decreased in the 300 mg/kg/d group from day 1 to 7, 24 to 28 and from day 35 to 38. However, a treatment relationship for the changes in water consumption is questionable.

In the functional observational battery (FOB) no treatment-related changes in autonomous, sensomotoric, neuromuscular, and central nervous system including body temperature measurements were noted. All motor activity parameters – number of counts, on-time, off-time, total distance, rearing, rearing time – did not show treatment-related differences of dose groups versus control.

Hematology and coagulation measurements showed no findings during the treatment and treatment-free recovery period.

Clinical chemistry evaluation revealed a slight increase of chloride (up to 1.06-fold) and cholesterol (up to 2.57-fold) in both sexes at 100 and 300 mg/kg/d at the end of the treatment period (day 29). Additionally, total protein was slightly (1.07-fold) increased in 100 mg/kg/d males and in both sexes at 300 mg/kg/d. An increase of bile acids was observed in 300 mg/kg/d males only. At the end of the treatment-free recovery period (day 43) a partial recovery of effects was observed. However, at 300 mg/kg/d bile acids (1.62-fold) and total protein (1.04-fold) were still slightly increased in males and chloride (1.02-fold) and cholesterol (1.41-fold) in females.

Urinalysis did not reveal any treatment-related effects.

At gross pathology, the main kill animals (day 29) showed a dose-dependent increase of liver

size in all dose groups. At recovery kill (day 43) an increased liver size was still detectable in the

300 mg/kg/d animals.

The organ weights of the main kill animals revealed a dose-dependent increase of liver weights in both sexes. In 30 mg/kg/d animals, only a trend (without statistical significance) towards increased liver weights, 25% in males and 9% in females, was seen. The absolute liver weight increase in males was about 58% in 100 mg/kg/d animals and 91% in 300 mg/kg/d animals. In females an absolute liver weight increase of about 42% was observed at 100 mg/kg/d and 68% at 300 mg/kg/d. Thyroid weights were increased in males at 100 and 300 mg/kg/d. The absolute thyroid weight increase achieved approximately 31% at 100 mg/kg/d and 72% at 300 mg/kg/d.

The observed liver and thyroid weight increases were not completely reversible within the 2- week treatment-free recovery period. At 300 mg/kg/d the liver weights were still increased by approximately 27% in males and 23% in females and the thyroid weights by approximately 51% in males.

Histopathology of the main kill animals revealed treatment-related lesions in the liver, thyroids and kidneys at all dose levels with a dose-dependent increase in incidence and severity.

At 30 mg/kg/d a minimal to mild centrilobular hepatocellular hypertrophy occurred in 3/5 males and 2/5 females. Minimal to mild follicular hyperplasia/hypertrophy of the thyroid gland was seen in 3/5 males and 2/5 females. In the proximal tubules of the kidney 3/5 males showed a minimal eosinophilic droplet accumulation.

At 100 mg/kg/d, a moderate centrilobular hepatocellular hypertrophy was detected in all animals.

In the thyroid gland, 4/5 males and all females showed a mild to moderate follicular hyperplasia/hypertrophy. A minimal condensation of colloid was seen in one male. In the kidney a mild to moderate accumulation of eosinophilic droplets within the proximal tubules was detected in 4/5 males.

At 300 mg/kg/d histopathology showed a marked centrilobular hepatocellular hypertrophy in all animals. One female exhibited mildly increased numbers of single cell necrosis in the

centrilobular area. A mild to marked follicular hyperplasia/hypertrophy of the thyroid gland was detected in all animals. However, the severity of this finding was higher in males. A minimal to mild condensation of colloid was seen in all males whereas females were unaffected. In the kidney, a mild to moderate accumulation of eosinophilic droplets in the tubular epithelium was observed in males only.

Histopathology of 300 mg/kg/d recovery kill animals showed a minimal to moderate centrilobular hepatocellular hypertrophy in all animals and increased numbers of single cell necrosis in the centrilobular area in 3/5 males with a minimal to mild severity. A minimal to

moderate follicular hyperplasia/ hypertrophy of the thyroid gland was detected in 4/5 males and 3/5 females. In the proximal tubules of the kidney a mild eosinophilic droplet accumulation was seen in all males.

 

Daily oral treatment with the test item at doses of 30, 100 and 300 mg/kg was tolerated over 4 weeks. In all dose groups histopathological investigations revealed centrilobular hepatocellular hypertrophy, follicular hyperplasia/ hypertrophy of the thyroid gland, condensation of colloid and accumulation of eosinophilic droplets in the tubular epithelium of the kidneys. All findings exhibited a clear dose-dependency with regard to incidence and severity and are not considered to represent adverse effects but rather reflect metabolic adaptive changes.

After the two week recovery period, the same findings were observed as in main kill animals, but with less pronounced severity. Therefore, only a tendency towards reversibility could be shown.

Centrilobular liver cell hypertrophy is an adaptive metabolic response after exposure to a microsomal enzyme inducing xenobiotic. The follicular hypertrophy/hyperplasia in the thyroid gland is a consequence of increased metabolism of thyroid hormones in the liver. It is well established that the rat is especially prone to this thyroid activation (Yoshizawa et al. 2007). The cause for the eosinophilic droplet accumulation in the kidney of male rats could not be elucidated in the current study.

The alterations of cholesterol, total protein and total bile acid may be considered as an indicator of increased metabolism and are in line with the findings seen in histopathology (hepatocellular hypertrophy). The reason for the dose-dependent increase in chloride is unclear, because it was not accompanied by further pathological alterations (e.g. imbalance of other serum electrolytes) and recovered nearly completely.

The No Observed Adverse Effect Level (NOAEL) of the test item in Wistar (Han) rats was established at 300 mg/kg/d.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and Guideline conform study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute toxiciy study (oral route)

A subacute toxicity study in rats according to OECD TG 407 was conducted with the test item. 

The test item was administered orally by gavage, once daily, 7 times a week, for 4 weeks to 3 groups of male and female Crl:WI (Han) rats at doses of 30, 100 or 300 mg/kg. A similarly constituted control group received the vehicle, 0.5% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium).

Daily oral treatment with the test item at doses of 30, 100 and 300 mg/kg was tolerated over 4 weeks. In all dose groups histopathological investigations revealed centrilobular hepatocellular hypertrophy, follicular hyperplasia/ hypertrophy of the thyroid gland, condensation of colloid and accumulation of eosinophilic droplets in the tubular epithelium of the kidneys. All findings exhibited a clear dose-dependency with regard to incidence and severity and are not considered to represent adverse effects but rather reflect metabolic adaptive changes.

After the two week recovery period, the same findings were observed as in main kill animals, but with less pronounced severity. Therefore, only a tendency towards reversibility could be shown.

Centrilobular liver cell hypertrophy is an adaptive metabolic response after exposure to a microsomal enzyme inducing xenobiotic. The follicular hypertrophy/hyperplasia in the thyroid gland is a consequence of increased metabolism of thyroid hormones in the liver. It is well established that the rat is especially prone to this thyroid activation (Yoshizawa et al. 2007). The cause for the eosinophilic droplet accumulation in the kidney of male rats could not be elucidated in the current study.

The alterations of cholesterol, total protein and total bile acid may be considered as an indicator of increased metabolism and are in line with the findings seen in histopathology (hepatocellular hypertrophy). The reason for the dose-dependent increase in chloride is unclear, because it was not accompanied by further pathological alterations (e.g. imbalance of other serum electrolytes) and recovered nearly completely.

The No Observed Adverse Effect Level (NOAEL) of the test item in Wistar (Han) rats was established at 300 mg/kg/d.

Justification for classification or non-classification

The available data on repeated dose toxicity are conclusive but not sufficient to meet the criteria for classification according to Regulation (EC) 1272/2008.