Propanoic acid, 3-[[bis(2-methylpropoxy)phosphinothioyl]thio]-2-methyl-

Endpoint summary

Description of key information

Repeated-dose toxicity was assessed in a GLP-compliant subacute and a GLP-compliant subchronic oral gavage toxicity study in rats. The subacute study design included a 14-day recovery element. The study designs and reporting details followed OECD testing guidelines 407 (1995) and 408. Both studies showed an adaptive liver enlargement at doses of 450 - 500 mg/kg bw. The NOEL was 125 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Mar 2014 - 03 Feb 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, avoid temp >30°C

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 +/- 1 days
- Weight at study initiation: 130g females and 160 g males
- Fasting period before study: no
- Housing: 5 animals per cage in H-Temp polysulfonate cages type 2000P
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 4 2014 To: June 13 2014

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently mixed with a magnetic stirrer. The test-substance preparations were produced at least weekly and stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Solubility and non-toxicity to rats
- Concentration in vehicle: adjusted to dose
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test material in corn oil at room temperature for a period of 7 days was proven before the start of the administration period. Homogeneous distribution of the test article in the test substance preparations was performed in the highest and lowest concentration. Additionally, concentration control was performed in all concentrations at the beginning of the administration period. Furthermore, each 2 samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study. The samples were analyzed only, if any imprecision while analysis of samples from start of the study occurs. All samples were stored until finalization of the report.

Duration of treatment / exposure:

92 days

Frequency of treatment:

daily

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the existing OECD 407 study

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working day and once daily on Saturday, Sunday and public holidays. If animals were in a moribund state, they were sacrificed and dissected.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were checked daily for any abnormal clinical signs prior administration, as well as, within 2 hours and 5 hours post administration. Detailed clinical observation (DCO) was performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmos, feces (appearance/ consistency) , urine , pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between body weight on the respective day of weighing and body weight on day 0 was calculated as body weight change.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At the end of the administration period, on study day 91.
- Dose groups that were examined: control and highest dose group animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the administration period, on study day 92.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the administration period, on study day 92.
- Animals fasted: Yes
- How many animals: 5 per sex and dose
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), g-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG), Bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: study day 85
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (For urinalysis the individual animals were transferred to metabolism cages
(withdrawal of food and water) and urine was collected overnight.)
- Parameters examined: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume,

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: days 86 - 91
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / (FOB)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix

HISTOPATHOLOGY: Yes (see table)

Statistics:

Blood parameters For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York Holm (1979): A Simple Sequentially Rejective Multiple Test Procedure. Scand. J. Statist. 6, 65-70

Urinalysis parameters (except pH, urine volume, specific gravity, color and turbidity Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York

Urine pH, volume, specific gravity, color and turbidity Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically. * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York

Weight parameters Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians * for p ≤ 0.05
** for p ≤-0.01 HETTMANNSPERGER, T.P. (1984): Statistical Inference based on Ranks, John W

Clinical signs:

no effects observed

Description (incidence and severity):

Salivation after treatment from slight to moderate was observed in all male and female animals of test group 3 (400 mg/kg bw/d) as well as in 7 of 10 male and 3 of 10 female animals of test group 2 (125 mg/kg bw/d). From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. No clinical findings were observed for male and female animals in test group 1 (25 mg/kg bw/d).

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related changes of mean body weights and mean body weight change values in both sexes were observed. For female animals of test group 1 (25 mg/kg bw/d) mean body weight change value was significantly higher on study day 49 (+15%). This single event was assessed as spontaneous in nature and not to be of toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were observed.
Female animal No. 49 of test group 0 (0 mg/kg bw/d) showed an unsharp iris frame at the left eye during the ophthalmological examination on study day 91. As the animal belonged to the control group a relation to treatment was excluded.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After three months of compound administration, hematocrit and hemoglobin values were decreased in rats of both sexes of test group 3 (400 mg/kg bw/d). Additionally, in females of the same test group red blood cell (RBC) counts were decreased. In male animals of test group 2 (125 mg/kg bw/d) hemoglobin values were also decreased, but the change was marginal (mean compared to controls was 2% lower) and this was the only altered measured red blood cell parameter. Therefore, this change was regarded as possibly treatment-related but not adverse (Mueller et al., 2006). In animals of both sexes of test group 2 and 3 (125 and 400 mg/kg bw/d) mean corpuscular volume (MCV) and in males of the mentioned test groups mean corpuscular hemoglobin (MCH) were decreased, but the changes were not dose-dependent and, therefore, they were regarded as incidental and not treatment-related. In males of test group 3 (400 mg/kg bw/d) prothrombin time (HQT = Hepatoquick’s test) was prolonged. In females of test groups 2 and 3 (125 and 400 mg/kg bw/d) prothrombin time
was shortened, but the values were within the historical control range. Therefore, this change was regarded as incidental and not treatmentrelated. In females of test group 3 (400 mg/kg bw/d) total white blood cell (WBC) counts and absolute lymphocyte counts were increased (WBC not statistically significantly). Absolute lymphocyte counts were already higher in females of test group 2 (125 mg/kg bw/d) and relative lymphocyte counts were increased and relative neutrophil counts decreased in females of test groups 1, 2 and 3 (25, 125 and 400 mg/kg bw/d). However, all mentioned values were within historical control ranges. Higher absolute and relative large unstained cell (LUC) counts in males of test group 2 (125 mg/kg bw/d) were not dose-dependently changed. Therefore, the in this paragraph mentioned alterations of the differential blood cell counts were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period in rats of both sexes of test group 3 (400 mg/kg bw/d) alkaline phosphatase (ALP) activities were increased and creatinine levels were decreased. Additionally, in males of this test group cholesterol values were decreased and potassium levels were increased and in females of the same test group glucose levels were higher compared to controls. Inorganic phosphate levels in females were higher compared to controls in test groups 1, 2 and 3 (25, 125 and 400 mg/kg bw/d). The means were not dose-dependently changed, but the medians of test groups 1 and 2 were within, that one of test groups 3 above the historical control range. Therefore, the changes in test groups 1 and 2 were regarded as incidental, but the change in test group 3 was assessed to be adverse. Total bilirubin levels were higher in rats of both sexes of test group 1 (25 mg/kg bw/d) and cholesterol values were increased in females of test group 2 (125 mg/kg bw/d), but both parameters were not dose-dependently changed and therefore the alterations were regarded as incidental and not treatment-related.

Urinalysis findings:

no effects observed

Description (incidence and severity):

In rats of both sexes of test groups 2 and 3 (125 and 400 mg/kg bw/d) ketone body levels in the urine were higher compared to controls. This was most probably a positive reaction of the semiquantitative test strip reaction to thiophosphate metabolites of the compound which were excreted via the kidneys. The keton body measurement of the test strip is based on the Legal’s test which gives a false positive reaction with compounds containing sulfhydryl groups (Combur-10 Test M test manual, Roche). Therefore, these results were regarded as treatment-related but not adverse. In male animals of test groups 2 and 3 (125 and 400 mg/kg bw/d) pH values were decreased and the incidences of granulated and epithelial cast in the urine were increased. In males of test group 3 (400 mg/kg bw/d) urine volumes and the incidence of transitional epithelial cells were increased; pH value decrease and higher urine volume per se were not regarded as adverse effects. The lower urine pH value may be due to the excretion of acid metabolites of the compound. The occurrence of granulated and epithelial casts and transitional epithelial cells in male rats of this age were most probably due to the α2u-globulinurie in these individuals which was observed histopathologically. The finding is regarded as a rat-specific effect without relevance for humans (Hard et al, 1993). In males of test groups 1 and 3 (25 and 400 mg/kg bw/d) specific gravity in the urine was lower compared to controls, but the change was not dose-dependent and therefore it was regarded as not treatment-related. In females of test group 3 (400 mg/kg bw/d) higher amounts of crystals with unknown origin were found in the urine and the pH value of the urine was lower compared to controls (not statistically significantly). The cause for the lower urine pH value was the same as described for male animals and was not regarded to be adverse.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental. The following examinations were performed during FOB and have to be assessed individually:
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed.
Motor activity measurement: No test substance-related changes were observed.
Comparing the single intervals of test substance-treated groups with the control group, isolated changes within single interval Nos. 2 and 3 were observed for female animals of test groups 1 and 2 (25 and 125 mg/kg bw/d). In addition, the overall motor activity was increased in female animals of test groups 1 and 2 (25 and 125 mg/kg bw/d). As no dose-response relationship occurred the changes were assessed to be incidental and not related to treatment. For female animals of test group 3 (400 mg/kg bw/d) increased value was detected for interval 3. The occurrence of this single change was assessed to be incidental and not related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative organ weight changed were observed for liver, kidney and pituitary gland. See table below for details. The increase in absolute and relative kidney weights in male animals of test groups 2 and 3 (125 and 400 mg/kg bw/d), the increase in absolute liver weight in males of test group 3 (400 mg/kg bw/d) and in relative liver weight in males of test group 2 and 3 (125 and 400 mg/kg bw/d) as well as the increase in absolute and relative female liver weight of test groups 2 and 3 (125 and 400 mg/kg bw/y) were regarded to be treatment-related. The increase in absolute and relative pituitary weight in females was regarded to be incidental and not related to treatment due to the missing histopathologic correlate and the fact that the increased pituitary weights were still within the historical control values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

All male and female animals of high dose group (400 mg/kg bw/d) revealed enlarged livers. In addition, three females of this test group showed a discoloration of the liver. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Treatment-related findings were observed in liver, kidneys, and thyroid glands. Animals of test group 3 (400 mg/kg bw/d) revealed a diffuse (males only) or centrilobular hypertrophy in the liver (males and females). In test group 2 (125 mg/kg bw/d) still one male and three females revealed a centrilobular hypertrophy. Three females of test group 3 (400 mg/kg bw/d) showed a diffuse storage of a light to middle brown pigment with centrilobular accentuation. This pigment was negative for Hall’s stain (bile), negative for Turnbull stain (iron) and negative for autofluorescence (lipofuscin). These findings were regarded to be treatment-related.
Males of test groups 2 and 3 (125 and 400 mg/kg bw/d) revealed eosinophilic droplets within the epithelial cells of the proximal tubules. These droplets were positive for the immunohistochemical stain against alpha 2u. This finding was regarded to be treatment-related.
In the thyroid gland of male and female animals of test groups 2 and 3 (125 and 400 mg/kg bw/d) hypertrophy and/or hyperplasia was observed. Two males of test group 3 (400 mg/kg bw/d) showed altered colloid in addition. These findings were regarded to be treatment-related.

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle: No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Sperm parameters: Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1: Absolute organ weights

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (25)	2 (125)	3 (400)	1 (25)	2 (125)	3 (400)
Kidneys	101%	107*%	122%**
Liver	103%	106%	136%**	106%	113%*	146%**
Pituitary gland				113%*	113%	119**%

*: p≤0.05, **: p≤0.01

Table 2: Relative Organ Weights

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (25)	2 (125)	3 (400)	1 (25)	2 (125)	3 (400)
Kidneys	100%	108%*	120%**	100%	105%	112%**
Liver	102%	106%*	134%**	101%	111%**	145%**
Pituitary gland				109%	112%	118%**

*: p≤0.05, **: p≤0.01

The increase in absolute and relative kidney weights in male animals of test groups 2 and 3 (125 and 400 mg/kg bw/d), the increase in absolute liver weight in males of test group 3 (400 mg/kg bw/d) and in relative liver weight in males of test group 2 and 3 (125 and 400 mg/kg bw/d) as well as the increase in absolute and relative female liver weight of test groups 2 and 3 (125 and 400 mg/k bw/d) were regarded to be treatment-related.

The increase in absolute and relative pituitary weight in females was regarded to be incidental and not related to treatment due to the missing histopathologic correlate and the fact that the increased pituitary weights were still within the historical control values.

Table 3: Histopathology

Liver	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (25)	2 (125)	3 (400)	0 (0)	1 (25)	2 (125)	3 (400)
No. of animals	10	10	10	10	10	10	10	10
Hypertrophy, diffuse	0	0	0	8	0	0	0	0
·        Grade 1				1
·        Grade 2				7
Hypertrophy, centrilobular	0	0	1	2	0	0	3	10
·        Grade 1			1	1			3	3
·        Grade 2				1				7
Pigment storage	0	0	0	0	0	0	0	3
·        Grade 1								2
·        Grade 2								1

Three females of test group 3 (400 mg/kg bw/d) showed a diffuse storage of a light to middle brown pigment with centrilobular accentuation. This pigment was negative for Hall’s stain (bile), negative for Turnbull stain (iron) and negative for autofluorescence (lipofuscin).

Kidneys	Male animals
Test group (mg/kg bw/d)	0 (0)	1 (25)	2 (125)	3 (400)
No. of animals	10	10	10	10
Eosinophilic droplets	0	0	4	4
·        Grade 1			3	2
·        Grade 2			1	2

Males of test groups 2 and 3 (125 and 400 mg/kg bw/d) revealed eosinophilic droplets within the epithelial cells of the proximal tubules. These droplets were positive for the immunohistochemical stain against alpha 2u. This finding was regarded to be treatment-related.

Thyroid glands	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (25)	2 (125)	3 (400)	0 (0)	1 (25)	2 (125)	3 (400)
No. of animals	10	10	10	10	10	10	10	10
Hypertr./hyperplasia	0	0	5	10	0	0	2	3
·        Grade 1			5	3			1
·        Grade 2				7			1	3
Altered colloid	1			2
·        Grade 1	1
·        Grade 2				2

In the thyroid gland of male and female animals of test groups 2 and 3 (125 and 400 mg/kg bw/d) hypertrophy and/or hyperplasia was observed. Two males of test group 3 (400 mg/kg bw/d) showed altered colloid in addition. Effects on thyroid are considered to be a rat-specific secondary response to the strong liver induction.

Conclusions:

In conclusion, the administration of test article by gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of systemic toxicity at a dose level of 400 mg/kg bw/d. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 125 mg/kg bw/d for male and female Wistar rats.

Executive summary:

In a GLP-compliant repeated dose toxicity study following OECD guideline 408, the test article was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 25 (test group 1), 125 (test group 2) and 400 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months. In addition to the required examinations special attention was given to the reproductive organs of male and female animals. Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. For at least 3 weeks an estrous cycle determination was performed. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations. Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals for sperm examinations.

In high dose animals, decreased hemoglobin and hematocrit values in both sexes were observed. Red blood cell (RBC) counts in females were decreased, prothrombin time (HQT) in males was prolonged. Increases in total white blood cell (WBC) counts and absolute lymphocyte counts in females were recorded. Alkaline phosphatase (ALP) activities were increased, creatinine values were decreased in both sexes. In males, cholesterol values were decreased and potassium levels were increased. In females, glucose and inorganic phosphate values in females were increased and an increased incidences of crystals of unknown origin was reported. Absolute and relative liver weights were increase in male (+36% and +34%, respectively) as well as female animals (+46% and +45%, respectively). Liver cell hypertrophy (diffuse or centrilobular) in males and females was observed. In animals of the low dose and intermediate dose level, no findings were reported. In conclusion, the administration of test article by gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of systemic toxicity at a dose level of 400 mg/kg bw/d. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 125 mg/kg bw/d for male and female Wistar rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP-compliant guideline study

System:

hepatobiliary

Organ:

liver

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 Toxicity Data

Repeated-dose toxicity was assessed in a GLP-compliant subacute oral gavage toxicity study including a 14-day recovery element using Wistar rats (2000). The study design and reporting details follow OECD testing guideline 407 (1995). The substance was administered at doses of 0 (vehicle, PEG 300), 20, 100 and 500 mg/kg/day. These doses were selected based on a 5-day range-finder study which showed changes in organ weight and food consumption at 600 and 1000 mg/kg bw. A later range finder study performed with female rats for 14 days and dose levels of 250, 500 and 1000 mg/kg bw showed that the highest dose caused mortality in 2/5 animals on day 1 and 2 after dosing (2011). Clinical symptoms were observed at this dose group. There were no effects on body weight. Further parameters were not examined. In contrast to the above study, corn oil instead of PEG 300 was used as vehicle.

Oral administration of the test item to Wistar rats at doses of 20, 100 and 500 mg/kg/day, for 28 days resulted in no effects upon mortality, clinical signs of toxicity, food consumption or body weight development, grip strength or locomotor activity, hematology parameters, macro- or microscopic changes at any dose level tested. Higher triglyceride and phospholipid levels noted in both sexes treated with 500 mg/kg/day, higher total cholesterol levels noted in females treated with 500 mg/kg/day and ketone in the urine of males and females treated with 100 mg/kg/day or 500 mg/kg/day were considered to be adaptive effects on lipid metabolism. These differences were reversible after the recovery period. Kidney weights were slightly increased at 100 mg/kg/day and 500 mg/kg/day, and liver weights were clearly increased at 500 mg/kg/day. The differences noted in the kidney weights of males and females treated with 500 mg/kg/day were reversible after recovery, as were the liver weight differences in females. Higher liver weights persisted in males after recovery, although clear indications of reversibility were seen. These changes were considered to be adaptive. Based on the results of this study, 20 mg/kg body weight/day of was established as the no-observed-effect-level (NOEL) and 500 mg/kg/day was considered to be the no-observed-adverse-effect-level (NOAEL).

 

Subchronic Toxicity Data

In a GLP-compliant repeated dose toxicity study following OECD guideline 408, the test article was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 25 (test group 1), 125 (test group 2) and 400 mg/kg bw/d (test group 3) over a period of 3 months (2015). In addition to the required examinations special attention was given to the reproductive organs of male and female animals. Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals for sperm examinations. Signs of general systemic toxicity were not observed even at a dose level of 400 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained. In male and female rats of test group 3 (400 mg/kg bw/d) a slight anemia was indicated by decreased hemoglobin, hematocrit and red blood cell (RBC) counts (only in females). In males of the mentioned test group an effect of the coagulation could be assumed by a prolonged prothrombin time and in females some chronic stress or inflammation was expressed by higher absolute lymphocyte counts. Liver cell swelling led to higher alkaline phosphatase activities in male and female rats of test group 3 (400 mg/kg bw/d) and the liver cell metabolism seemed to be affected because of lower cholesterol levels in males and higher glucose levels in females of this test group. Lower serum creatinine levels may be due to a decreased synthesis of creatin as precursor of creatinine in the liver cells. An increased excretion of creatinine via the kidneys may also be possible. In the livers of male and female animals in test groups 2 and 3 (125 and 400 mg/kg bw/d) a diffuse or centrilobular hypertrophy in a dose-depended manner was observed. This type of finding also corresponded to the liver weight increase in this test groups. Females of test group 3 (400 mg/kg bw/d) revealed in addition a light to middle brown pigment in the liver. The character of the pigment could not further be determined, but it was negative for special stains for iron, bile and lipofuscin. In combination with deviations in clinical chemistry the above mentioned findings were regarded to be treatment-related and adverse for animals of test group 3 (400 mg/kg bw/d). In test group 2 (125 mg/kg bw/d) there was still an increase in liver weight and liver hypertrophy was observed in some males and females. Due to the fact that there were no other additional histopathologic findings and not more than one clinical chemistry parameter was changed, these findings were regarded to be treatment-related but not adverse in nature (Hall et al., 2012).

As a possible secondary effect to the liver hypertrophy, males and females of test groups 2 and 3 (125 and 400 mg/kg bw/d) showed a hypertrophy/hyperplasia in the thyroid gland. This could have been caused by the liver cell hypertrophy (most likely enzyme induction). It was regarded to be a secondary finding, treatment-related but not relevant for humans. In the kidneys of male animals in test groups 2 and 3 (125 and 400 mg/kg bw/d) eosinophilic droplets were observed. These droplets were immunohistologically stained positive for alpha 2u globulin. Eosinophilic droplets in the proximal convoluted tubules represent alpha 2u globulin, a poorly hydrolysable, low molecular weight protein characteristic for male rats. The increase of eosinophilic droplets was regarded to be treatment-related, but is not of human relevance. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. In conclusion, the administration of the test article by gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of systemic toxicity at a dose level of 400 mg/kg bw/d. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 125 mg/kg bw/d for male and female Wistar rats.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The NOAEL for subchronic exposure was established at 125 mg/kg/day based on liver weight increases at 400 mg/kg/day. As a result the substance is not considered to be classified for repeated dose toxicity via oral route under Regulation (EC) No 1272/2008.