1-[5-(2,6-difluorophenyl)-4,5-dihydro-1,2-oxazol-3-yl]ethanone

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF Japan, 2-1-9, 90-Day Repeated Oral Toxicity Studies

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Purity: 89.7% by analysis

Test animals

Species:

rat

Strain:

other: Hsd: Sprague Dawley SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 49 – 55 days
- Weight at study initiation: Males: 144.61 to 175.56 g; Females: 114.18 to 140.13 g
- Housing: two per sex per cage in clean, sterilized polycarbonate rat cages (size: approximately 41.0 L x 26.5 W x 20.0 H cm) covered with stainless steel grill tops. Enrichment items (nestlings) were placed in each cage. Autoclaved corncob was used as the bedding.
- Diet (e.g. ad libitum): Nutrilab Rodent Mash feed, ad libitum
- Water (e.g. ad libitum): Reverse osmosis-treated (on-site) drinking water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 24.4°C
- Humidity (%): 39 to 68%
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): once or twice in a two week period and used within the stability period (22 days) of the test item in the diet.
- Mixing appropriate amounts with (Type of food): Prior to the experimental diet preparation, the test item was ground using a mortar and pestle. After grinding, the test item was passed through a sieve (~75 micron). The sieved particles were collected in a bottom pan. The required amount of the test item was weighed and added to approximately 1/10th of the quantity of the total feed needed to prepare a premix. Premix feed was mixed in an electric mixer. Premixed feed was homogeneously mixed with the remaining quantity of feed in a blender for approximately 20 minutes.
- Storage temperature of food: ambient (20 to 25°C)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose confirmation and homogeneity analysis was performed on day 0 (one day prior the initiation of treatment) and on days 49, and 85 of treatment at 120, 700, 4200, and 14000 ppm concentration levels in the diet. The analysis was carried out using an HPLC column equipped with UV/DAD detector. The active ingredient content (±20%) and homogeneity (% RSD: <15) was found to be within the acceptable limits in the diet. Test item was not detected in control diet samples.

Duration of treatment / exposure:

90 days males, 91 days females

Frequency of treatment:

daily in the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

120 ppm

Doses / concentrations 2

Dose / conc.:

700 ppm

Doses / concentrations 3

Dose / conc.:

4 200 ppm

Doses / concentrations 4

Dose / conc.:

14 000 ppm

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dosage levels were selected based on a 14-day dose range finding study and in consultation with the Sponsor. The high concentration, 14000 ppm, was expected to produce minimal toxicity. The other concentrations were selected to assess a dose response for any observed effects and to establish a no-observed-adverse-effect-level (NOAEL).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed once daily at the same time each day (±2 hours). Detailed clinical examinations were performed for all animals on Day 1 (prior to the initiation of treatment) and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment prior to exposure and weekly thereafter.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to initiation of treatment and during the last week of treatment (week 13).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table No. 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. At the end of treatment period, all the animals were euthanized in stratified order by exsanguination under deep anaesthesia with isoflurane. All animals were examined for gross pathological changes.
HISTOPATHOLOGY: Yes. See Table 3 for organs collected, weighed, and preserved. Adherent adipose tissue from the organs was removed and the organ weights were recorded. Paired organs were weighed together. Terminal body weights on the day of necropsy were used to calculate organ weights relative to body weight. Organ weights relative to brain weight was also calculated. Organs were preserved in 10% neutral buffered formalin except the testes, which, were preserved in modified Davidson's fixative, and the epididymides and eyes were preserved in Davidson's fixative, both for 48 hours and were then transferred to 10% neutral buffered formalin. Histopathologic examinations were performed on all preserved organs of the control and high dose group (14000 ppm). In addition, histopathological evaluation of the liver, sternum with bone marrow, thyroid, and teeth in both sexes as well as the pituitary in males was performed in the low, intermediate, and high intermediate dose groups, as they were identified as target organs based on findings in the high dose group. To rule out the possibility of being a target, the prostate was also microscopically examined in the lower dose groups. Additionally, the teeth were identified as a potential target organ by the peer review pathologist, and hence it was also processed in low, mid and high intermediate dose groups. Gross lesions observed in 120, 700, and 14000 ppm females were also read microscopically. The tissues were processed and embedded in paraffin, sectioned, and stained with haematoxylin and eosin.

Statistics:

Statistical analysis was performed using validated statistical software (Graph Pad Prism 5.02). Analyses were conducted using two-tailed tests for a minimum significance level of 5%, comparing each test item treated group to the control group for each sex. Each mean was presented with the standard deviation (SD) and the number of animals (N) used to calculate the mean. A 'p' value ≤ 0.05 was considered as statistically significant.
Numerical data, including body weights, body weight gain, food consumption, clinical pathology (haematology, clinical chemistry, and urinalysis) and organ weights were subjected to a statistical analysis. When an individual observation was recorded as being less than a certain value, calculations were performed on half the recorded value. When an individual observation was recorded as being greater than a certain value, calculations were performed on the recorded value. The data was examined for outliers and when required was subjected to Grubb's test. The significant values in Grubb's test were excluded from statistical analysis. Initially data was subjected to a Shapiro-Wilk normality test to check for normal distribution of the data and Bartlett's test for homogeneity of variance. When Bartlett's test or the Shapiro-Wilk normality tests were non-significant, Dunnett‘s post hoc test was used to compare the control group with test item treated groups. When Bartlett's test or Shapiro-Wilk normality tests were significant, data was subjected to a Kruskal-Wallis ANOVA followed by Dunn‘s post hoc test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed in either males or females at any concentration.

Mortality:

no mortality observed

Description (incidence):

There was no mortality observed in either males or females at any concentration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item-related effects were noted in body weight parameters in high dose (14000 ppm) males (adverse) and females (nonadverse). A statistically significant reduction in mean body weight in relation to the control group was observed from week 1 to 13 in males (8-12%) and from week 4 to 13 in females (6-9%) at 14000 ppm. The final (Day 90) body weights in males and females at 14000 ppm were 12% and 8% lower than controls, respectively. Statistically significant decreases in body weight gains were observed for the weeks 0-1, 6-7, 8-9, 12-13, 0-4, 4-8, 8-13, for the overall interval (week 1-13) in males and for the weeks 0-4, and 1-13 in females in the 14000 ppm group. Overall mean body weight gains (week 1-13) in males and females at 14000 ppm were 20% and 17% lower than controls, respectively. The changes in the body weight parameters at 14000 ppm group were considered test item related in both sexes and adverse in males. The changes noted in females were not considered adverse because the body weight decreases were <10% and there were no statistically significant decreases in food efficiency compared to controls. There were no test item-related changes in body weight parameters at <4200 ppm in both sexes. The sporadic statistical significance noted in body weight gains at the lower doses were not considered to be test item related due to the minimal nature of the changes and the lack of a dose response.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Overall (Weeks 1-13) test item intakes were 9, 47, 279, and 965 mg/kg body weight/day in males and 10, 57, 313, and 1140 mg/kg bw/day in females, at the 120, 700, 4200, and 14000 ppm concentration levels, respectively. Test item-related effects were noted in food consumption in males and females at 14000 ppm. A statistically significant reduction in food consumption in relation to the control group was observed at weeks 0-1, 1-2, 4-5, 6-7, 8-9, 11-12, 12-13, 0-4, and 8-13 in males and 0-1, 1-2, 2-3, 6-7, 9-10, 10-11, 0-4, and 4-8 in females in the 14000 ppm group. Overall (week 1-13) food consumption in males and females at 14000 ppm was 8% (statistically significant) and 4%, lower as compared to controls, respectively. The reduced food consumption correlated with changes in body weight parameters at 14000 ppm and was considered test item related. Since changes in overall food consumption (week 1-13) were minimal (<8%) in both sexes, they were not considered adverse. There were no test item related changes in food consumption at <4200 ppm in either sex. Statistically significant changes noted in food consumption at these levels were not considered test item-related, as they were sporadic, minimal, and not dose or time dependent.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Test item-related effects were noted in food efficiency (adverse in males) in males and females at 14000 ppm. A statistically significant reduction in food conversion efficiency in relation to controls was observed at weeks 0-1, 8-9, 12-13, and 8-13 in males and at week 3 in females in the 14000 ppm group. Overall (week 1-13) food efficiency in males and females at 14000 ppm were 28% (statistically significant) and 7% (statistically not significant) lower than controls, respectively. The food conversion efficiency changes correlated with changes in body weight parameters and were considered test item-related. While decreases in males were considered adverse, decreases in females were not considered adverse due to minimal nature of change.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmological lesions were observed in males or females at any concentration.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse changes in haematology or coagulation parameters in male or female rats at any concentration. A test item-related, but non-adverse decrease in reticulocyte count was observed in males at 14000 ppm. A statistically significant, but minimal decrease (-16%) in reticulocytes was observed in males at the 14000 ppm concentration. The value is within historical control range (187.02 – 268.88 109/L). This decrease was not associated with changes in red cell mass parameters (RBC, HGB, HCT), and a similar change in reticulocytes was not observed in females. In addition, there were no corresponding findings observed in the bone marrow histology of the males. Therefore, the minimal decrease in reticulocytes in the 14000 ppm male group may have been test item related, but was considered to be non-adverse.
All other statistically significant differences in haematology parameters were considered spurious and non-adverse. Group mean eosinophils in males fed 14000 ppm were statistically lower (-35%) compared to controls. However, this difference was not associated with changes in other white cell parameters and a similar change was not observed in females. Therefore, lower eosiniophils in the 14000 ppm male group was considered spurious and non-adverse. Group mean prothrombin time was lower in females fed 14000 ppm (-6% compared to control). This difference was not associated with changes in other coagulation parameters and a similar finding was not observed in male rats. In addition, the direction of change (decreased rather than increased) is not biologically relevant. Therefore, this finding was also considered spurious and non-adverse. There were no statistically significant changes in other haematology or coagulation parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse changes in clinical chemistry parameters in male or female rats at any concentration. Statistically significant changes in some liver-related clinical chemistry parameters, including selected liver enzymes and serum lipids and proteins, were present in some treated male and female groups. Based on the dose-response for these changes and the consistency of the changes between males and females, they were considered to be test item related, and were likely associated with liver enzyme induction. Induction of liver enzymes, a physiological response to the metabolism of a test item, was suggested by increased liver weight and associated microscopic hepatocellular hypertrophy at 4200 ppm in males and at 14000 ppm in both sexes. While these changes in liver-related parameters were considered test item related, they were not considered adverse based on the lack of correlative changes in other clinical or anatomic pathology parameters indicative of liver toxicity. ALT was statistically significantly decreased at 4200 and 14000 ppm in males and females. Similar changes were observed in a 14-day study in male and female rats fed the test item at >300 ppm. Therefore, the changes in ALT were considered test item related. ALT generally increases rather than decreases in association with adverse effects on the liver. Decreases (and increases) in ALT can be associated with enzyme induction. Therefore, the decreases in ALT were considered test item related, but non-adverse.
GGT was statistically significantly increased at 14000 ppm in males and females. The induction of hepatic GGT has been reported in conjunction with increased liver weight and centrilobular hypertrophy. In the absence of biologically significant increases in ALT or ALP, or adverse microscopic changes in the liver, the increases in GGT were considered test item related, but non-adverse. Total bilirubin was significantly decreased in females fed 14000 ppm (-18%) of test item. Total bilirubin can be reduced as a consequence of higher conjugation rates and excretion via bile when hepatic enzyme–inducing chemicals are administered. Hence, in absence of correlated microscopic findings, the minimal decrease in total bilirubin was considered test item related, but not adverse. Cholesterol was minimally increased in males and females at 14000 ppm (22% each sex and statistically significant). A statistically non-significant and mild increase in triglycerides was observed in males at 14000 ppm (38%). Mild increases in cholesterol levels have been observed to be associated with hepatocellular hypertrophy due to hepatic enzyme induction in rats. Lipid levels can be decreased or increased during enzyme induction depending on the compound administered and can also reflect changes in food consumption (the highest-dosed rats had lower body weights and food consumption). The increase in cholesterol and triglycerides was considered test item related and non-adverse due to the minimal nature of these increases. Total protein, albumin and globulin were significantly, but minimally, increased in males (but not females) at the highest concentration (10%, each parameter). A common cause of increased protein (albumin and globulin being a portion of the total protein) is decreased hydration associated with decreased food intake. And again, administration of enzyme inducers may also alter protein levels—the change often being correlated with changes in lipid levels, as is the case with this compound. The minimal increase in protein, albumin and globulin was considered test item related, but non-adverse. Calcium was very slightly elevated (5%) at the highest dose in males, but not in females. This slight elevation may reflect the elevation in total protein in this group, as much of calcium is protein-bound. This is a physiologically appropriate change and should not be considered adverse. ALP was statistically significantly increased only in males at 120 ppm (29%) and creatinine was statistically significantly decreased only in males at 4200 ppm (-10%). As there was not clear dose response, nor a histological correlate in these cases, the mild changes in ALP and creatinine were considered spurious. There were no statistically significant changes in other clinical chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no adverse changes in urinalysis parameters in male or female rats at any concentration.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Primary, test item-related, non-adverse organ weight changes were present in the liver of male and female rats fed 4200 and 14000 ppm of test item. In addition, non-adverse organ weight changes occurring secondary to body weight decrements were present in various organs at 14000 ppm. Terminal body weights after fasting were significantly lower in males (-13%) and females (-11%) at the 14000 ppm dose level as compared to the control group. Mean absolute and relative (to body and brain) liver weights were statistically significantly increased in rats fed 4200 and 14000 ppm of test item in both sexes (mean absolute and relative to brain liver weight increases were not significant for 4200 ppm females). Mean liver weight relative to body weight was increased 39% above controls in both 14000 ppm males and females, and 17% and 10% above controls in males and females, at 4200 ppm, respectively. Increased liver weight was correlated with increased hepatocellular hypertrophy at 4200 ppm in males and at 14000 ppm in both males and females. The test item associated liver weight increases were interpreted to be the result of induction of metabolic enzymes and non-adverse. The absolute and relative (to brain) weights of prostate, seminal vesicles and coagulating glands were significantly lower (24 and 20% compared to controls, respectively) in males treated with 14000 ppm of test item. These weight changes were not associated with correlative microscopic changes and were considered to be secondary to effects on body weight and food consumption at this dietary concentration. Decrements in body weight parameters are associated with decreases in accessory sex organ weights. Day 90 body weights and overall food consumption were decreased 12% and 8%, in the 14000 ppm male group, respectively. Statistically significant weight changes were observed in brain, heart, kidneys in males and females, as well as in the spleen and thymus in females, and testes and epididymides in males. These changes reflected test item-related decrements in body weight and food consumption in rats at 14000 ppm. Thus, in those organs that are typically less sensitive to body weight changes (brain, kidney, testes), the pattern of change in organ weight parameters was characterized by increased organ weight relative to body weight and minimal or no effects on absolute organ weight and organ weight relative to brain weight. For body weight-sensitive organs (spleen, thymus, heart) changes followed the general pattern of decreased absolute organ weight and organ weight relative to brain weight, with little or no change in organ weight relative to body weight. Statistically significant findings that lacked a clear dose response and were not consistent across all weight parameters were present in brain weight relative to body weight in male rats fed 120 ppm and epididymis weight relative to body weight in males fed 14000 ppm. None of these changes were correlated with microscopic findings and none were considered adverse.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related gross pathology findings observed in this study. All findings observed were incidental in nature.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Centrilobular hepatocellular hypertrophy was observed in rats fed 4200 ppm (males: 5/10) and 14000 ppm (males: 8/10, females: 8/10) of the test item. Hepatocellular hypertrophy was characterized by cytoplasmic enlargement of centrilobular hepatocytes with granular cytoplasm. This microscopic observation was correlated with an increase in liver weight parameters in these dose groups. As this observation is consistent with the induction of hepatic enzymes and was not associated with alterations in clinical pathology parameters indicative of liver toxicity, it was considered test item related, adaptive and non-adverse. Minimal to mild hepatocellular vacuolation was observed in both sexes at 14000 ppm (males: 3/10, females: 1/10). The affected hepatocytes were enlarged, contained microvesicles and were randomly distributed within the lobules with a pattern and morphology more consistent with organelle distension than lipid retention. This observation was not associated with adverse alterations in clinical chemistry parameters and may be associated with enzyme induction; it was considered test item related and potentially adverse. Minimal, focal hepatocellular necrosis was observed in 2/10 males at 14000 ppm. Considering the low incidence, minimal severity and lack of any lobular pattern, this change was unlikely related to test item administration.
Follicular cell hypertrophy was observed in all animals of both sexes at 14000 ppm. This change was also observed in 7/10 males at 4200 ppm. Follicular cell hypertrophy was characterized by the enlargement of follicular epithelial cells often associated with a reduction in follicular lumen and colloid content. This change may indicate altered thyroid gland homeostasis consistent with increased metabolism and elimination of thyroid hormones by induction of hepatocellular micosomal enzymes. This finding is consistent with the observed centrilobular hepatocellular hypertrophy at these doses and was considered potentially adverse for rats. Due to differences in T4 half-life, thyroglobulin binding, and the ease of UDP-glucuronyl-transferase induction, rats, compared to humans, are more sensitive to thyroid hormone depletion, as seen during hepatic enzyme induction, and have higher thyroid follicular cell proliferation rates. Thus, rats are much more susceptible than humans to secondary thyroid follicular cell hypertrophy. This effect is potentially adverse in rats, but not relevant to other species. Follicular cell hypertrophy also occurred in isolated cases in a control male and in one male at 700 ppm, but since these findings can occur as a normal background changes, these cases were considered unrelated to test item treatment.
Individual cell hypertrophy in cells of the pars distalis of the pituitary was observed in males at 4200 ppm (3/10) and 14000 ppm (5/10). Individual cell hypertrophy was characterized by the enlargement of scattered basophilic cells (presumably thyrotrophs) in the pars distalis. Similar to the changes observed in the thyroid in male rats at these doses, this observation indicates altered thyroid gland homeostasis. Decreased circulating thyroid hormones trigger an increase in the release of pituitary-derived thyroid stimulating hormone (TSH). Although individual cell hypertrophy in the pars distalis is consistent with an adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction, this finding was regarded as potentially adverse to rats. Males are slightly more sensitive than females to the perturbation in the hypothalamic–pituitary–thyroid axis, which would be consistent with the current findings.
Minimal decrease in bone marrow cellularity was observed in 5/10 females at 14000 ppm. Based on its minimal nature and the lack of an effect on red cell mass parameters (RBC, HGB, and HCT) or white blood cell parameters in peripheral blood, this finding was considered non-adverse and possibly related to the decrements in body weight and food consumption in this group.
Ameloblast degeneration (usually of minimal severity) was observed in the upper incisor teeth (Level I of the nose) of males and females from all treated groups and in male controls. The lesion occurred without a clear dose response across the treated groups and was not associated with any other microscopic or clinically observed changes in the teeth. Therefore, this change was considered to most likely represent a spurious finding. However, based upon a slight increase in the incidence and severity of ameloblast degeneration in males at 14000 ppm, a relationship to treatment could not be ruled out for males at this dietary concentration.
All other microscopic findings observed in this study were either spontaneous or incidental in nature as lesions were distributed randomly across the experimental groups.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All other microscopic findings observed in this study were either spontaneous or incidental in nature as lesions were distributed randomly across the experimental groups.

Other effects:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

700 other: ppm (equivalent to 47 mg/kg bw/day)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

4 200 other: ppm (equivalent to 313 mg/kg/day)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

1

Critical effects observed:

no

Lowest effective dose / conc.:

4 200 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Relevant for humans:

no

2

Critical effects observed:

no

Lowest effective dose / conc.:

4 200 ppm

System:

endocrine system

Organ:

pituitary gland

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Male NOAEL = 700 ppm (equivalent to 47 mg/kg bw/day)
Female NOAEL = 4200 ppm (equivalent to 313 mg/kg bw/day)

Executive summary:

Groups of 10 male and 10 female rats received daily oral dietary concentrations of 0, 120, 700, 4200, or 14000 ppm of the test item for a minimum 90 consecutive days (90 days for males and 91 days for females). The test item was shown to be homogeneous and at the targeted concentrations in the diet. The test item was shown to be stable for 22 days at room temperature in the diet prior to the study start. Parameters evaluated in this study included twice daily mortality checks, daily checks for clinical signs, weekly detailed clinical examinations, weekly measurement of body weight and food consumption and ophthalmological examinations before the start and at the end of the treatment period. At the end of the treatment period, haematology, clinical chemistry, urinalysis, gross pathology and organ weights were evaluated. All organs preserved from the control and high dose groups, all gross lesions and liver, sternum with bone marrow, thyroid, and teeth in both sexes and pituitary and prostate in males at intermediate concentrations were subjected to histopathology examinations. Overall (Weeks 1-13) test item intakes were 9, 47, 279, and 965 mg/kg body weight/day in males and 10, 57, 313, and 1140 mg/kg bw/day in females, at the 120, 700, 4200, and 14000 ppm concentration levels, respectively.

No mortality, clinical signs, or ophthalmological lesions were observed in males or females at any concentration.

Test item-related effects were noted in body weight and food intake parameters in males (adverse) and females (non-adverse) at 14000 ppm. The final (Day 90) body weights in males and females at this concentration were 12% and 8%, lower (statistically significant in both sexes) compared to controls, respectively. Overall (Weeks 1-13) body weight gains in males and females were 20% and 17% (statistically significant in both sexes), lower compared to controls, respectively. Overall (Weeks 1-13) food consumption in males and females were reduced by ≤ 10% at the 14000 ppm concentration. Overall (Weeks 1-13) food conversion efficiency in males and females was 28% and 7% lower than controls, respectively.

There were no adverse changes in haematology, coagulation, clinical chemistry, and urinalysis parameters. Non-adverse test item-related changes included decreased reticulocyte counts in males at 14000 ppm, decreased ALT at 4200 and 14000 ppm in males and females, increased GGT in males and females at 14000 ppm, increased cholesterol in males and females at 14000 ppm, increased triglycerides in males at 14000 ppm, increased total protein, albumin, globulin, and calcium in males at 14000 ppm, and decreased total bilirubin in females at 14000 ppm. Based on the statistical significance, the observation of dose-responses for these changes, and/or the consistency of the changes between males and females, these changes were considered to be test item related, and were likely associated with metabolic enzyme induction.

There were no test item related gross pathology findings observed in males or females. Test item-related increases in mean absolute and relative (to body and brain weight) liver weights were noted in the 4200 ppm males and 14000 ppm male and female groups. The test item associated liver weight increases were interpreted to be the result of induction of metabolic enzymes and non-adverse.

Test item related potentially adverse histopathological findings included thyroid follicular cell hypertrophy (>4200 ppm in males and at 14000 ppm in females), individual cell hypertrophy of the pars distalis of the pituitary (>4200 ppm, males only), and hepatocellular vacuolation (14000 ppm in males and females). Test item related, non-adverse findings in male and female rats included minimal centrilobular hepatocellular hypertrophy (>4200 ppm in males and at 14000 ppm in females), and minimally decreased cellularity of the bone marrow (14000 ppm, females only).

Under the conditions of the study, the No-Observed-Adverse-Effect-Level (NOAEL) of the test item in the male Sprague Dawley rats was 700 ppm, which is equivalent to 47 mg/kg bw/day. This NOAEL is based on thyroid follicular cell hypertrophy and pituitary pars distalis individual cell hypertrophy at 4200 ppm (279 mg/kg bw/day) and above. The NOAEL for the female rats was 4200 ppm, which is equivalent to 313 mg/kg bw/day. This NOAEL was based on hepatocellular vacuolation, and thyroid follicular cell hypertrophy at 14000 ppm (1140 mg/kg bw/day). The thyroid follicular cell hypertrophy is potentially adverse in rats, but not relevant to humans.