2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Endpoint summary

Description of key information

subacute toxicity:

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

subchronic toxicity:

OECD 408, oral, rat, 90-days: NOAEL = 259 mg/kg bw/day (males) and 902 mg/kg bw/day (females)

OECD 409, oral, dog, 90-days: LOAEL = 0.564 mg/kg bw/day (males) and 0.591 mg/kg bw/day (females); NOAEL < 0.564 mg/kg bw/day (males) and < 0.591 mg/kg bw/day (females)

chronic toxicity:

OECD 453, oral, rat, 24 months: NOAEL = 0.08 mg/kg bw/day (males) and 0.11 mg/kg bw/day (females)

OECD 452, oral, dog, 1-year: NOAEL = 0.102 mg/kg bw/day (males and females)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Jan - 31 July 2002

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)

Version / remarks:

adopted 21 Sep1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

Rats were housed individually instead of in groups per sex. Phytoestrogen levels were not determined in the diet. T4, T3 and TSH levels were not assessed. No vaginal smears taken for oestrus cycle assessment.

GLP compliance:

yes (incl. QA statement)

Remarks:

Secrétariat du Groupe Interminiseriel des Produits Chimiques, Paris, France

Limit test:

no

Species:

rat

Strain:

other: RJ: WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 6 weeks
- Weight at study initiation: 196 - 227 g (males), 158 - 186 g (females)
- Housing: individually in suspended stainless steel wire mesh cages
- Diet: certified rodent powdered and irradiated diet A04C-10 P1 (Usine d'Alimentation Rationnelle, Villemoisson-sur-Orge, France), ad libitum
- Water: filtered and softened water from municipal water supply, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 -24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: The test substance formulations were prepared approximately every three weeks. There were four preparations.
- Storage temperature of food: diet formulations were stored at below -15 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the test substance in the diet was verified on the first formulation at the lowest and highest concentrations to demonstrate adequate formulation procedures. Dietary levels of the test substance were verified for each concentration from all four diet preparations. All results for homogeneity and concentration were within a range of 90 to 106% of the nominal concentration. Therefore, all values were within the target range of 85 to 115% of the nominal concentration. The test substance was found to be stable in the rodent diet at concentrations of 1.25 ppm and 15000 ppm after 59 days at below -15 °C and 11 days at ambient temperature. The formulation at 15000 ppm was not administered and was prepared only to check the stability of the test substance in ground rodent diet. Hence the test material was stable in the diet over the period of usage on this study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously (via diet)

Dose / conc.:

1.25 ppm

Remarks:

equivalent to 0.08 and 0.09 mg/kg bw/day in males and females, respectively

Dose / conc.:

600 ppm

Remarks:

equivalent to 39.0 and 45.6 mg/kg bw/day in males and females, respectively

Dose / conc.:

4 000 ppm

Remarks:

equivalent to 259 and 302 mg/kg bw/day in males and females, respectively

Dose / conc.:

12 000 ppm

Remarks:

equivalent to 787 and 902 mg/kg bw/day in males and females, respectively

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were set after evaluation of a 14-day gavage study in the rat (Kennel, report no. SA 01134, 2001) with the test substance, where slight toxicity was observed at 600 mg/kg bw/day equivalent to approximately 6000 ppm. A low dose of 1.25 ppm was included in this 90-day study to ensure that a No Observed Effect level (NOEL) was achieved in terms of possible eye lesions.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends or public holidays)
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observed clinical signs were recorded at least once daily for all animals. Detailed physical examinations were performed during the acclimatization phase and at least weekly during the treatment period. The nature, onset, severity, reversibility and duration of clinical signs were recorded. Cages and cage-trays were inspected daily for evidence of ill-health, such as blood or loose feces.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded twice during the acclimatization period, on the first day of test substance administration, then at weekly intervals throughout the treatment period and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied and of that remaining at the end of the food consumption period was recorded weekly for all animals during the treatment period. The weekly mean achieved dosage intake in mg/kg bw/day for each week and for Weeks 1 to 13 was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimatization phase, all animals were subjected to an ophthalmological examination. After instillation of an atropinic agent (Mydriaticum, Merck Sharp & Dohme), each eye was examined by means of an indirect ophthalmoscope. During Week 3, 8 and 12, all surviving animals were re-examined.
- Dose groups that were examined: 1.25, 600, 4000 and 12000 ppm

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Days 85, 86 or 87, blood samples were taken from all surviving animals in all groups by puncture of the retro-orbital venous plexus
- Anaesthetic used for blood collection: Yes (inhalation of isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count and differential count evaluation and platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Days 85, 86 or 87, blood samples were taken from all surviving animals in all groups by puncture of the retro-orbital venous plexus
- Animals fasted: Yes
- How many animals: all
- Parameters examined: total bilirubin, glucose, urea, creatine, total cholesterol, triglycerides, chloride, sodium, potassium, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyltransferase, total protein, albumin, globulin and albumin/globulin ratio values were calculated

URINALYSIS: Yes
- Time schedule for collection of urine: overnight urine samples were collected on study Days 91, 92, 93 or 94, in the morning, prior to sacrifice
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Yes
- Parameters examined: any significant change in the general appearance, urinary volume, pH, urinary refractive index, glucose, bilirubin, ketone bodies, occult blood, protein, urobilinogen, microscopic examination (presence of red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during acclimatization phase and during Week 12
- Dose groups that were examined: 1.25, 600, 4000 and 12000 ppm
- Battery of functions tested: grasping reflex, righting reflex, corneal reflex, pupillary reflex, auditory startle reflex, head shaking reflex

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On Days 91, 92, 93 or 94, all surviving animals from all groups were necropsied. An approximately equal number of animals randomly distributed amongst all groups were sampled on each day. All animals, either found dead or killed by design, were necropsied. The necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded, sampled and examined microscopically.

The following organs or tissues were sampled: adrenal gland, aorta, articular surface (femorotibial), bone (sternum), bone marrow (sternum), brain, epididymis, esophagus, exorbital (lacrymal) gland, eye and optic nerve, harderian (lacrymal) gland, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidney, larynx/pharynx, liver, lung, lymph nodes (submaxillary, mesenteric), mammary gland, nasal cavities, ovary, pancreas, pituitary gland, prostate, sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, submaxillary (salivary) gland, testis, thymus, thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus (with cervix), vagina

A bone marrow smear was prepared from one femur, stained with May-Grünwald Giemsa, but not examined.
Samples were fixed by immersion in neutral buffered 10% formalin with the exception of the eye, optic nerve, harderian gland, epididymis and testis that were fixed in Davidson's fixative.
With the exception of larynx/pharynx and exorbital (lacrymal) gland, all the above mentioned organs and tissues were embedded in paraffin wax.

HISTOPATHOLOGY: Yes
Histological sections, stained with hematoxylin and eosin, were prepared for all the organs from all the animals in the control and high dose groups. Additionally, sections from the liver, lung, kidney, thyroid gland and pancreas and from significant gross findings observed at necropsy were prepared for all the animals in all intermediate dose groups. Histopathological examinations were performed on all the tissues from all the animals in the control and high dose groups. The liver, lung, thyroid gland and kidney were examined in all the animals in the study. Significant macroscopic findings were also examined in all dose groups.

Statistics:

Mean and standard deviation were calculated for each group and per time period for body weight change and average food consumption. All calculations and statistical analysis were performed using a dedicated computer system (Path/Tox System, version 4.2.2).
In general, Bartlett test was performed to compare the homogeneity of group variances.
If the Bartlett test was not significant (α = 0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (α = 0.05), the statistical procedure was stopped and group means were considered to be homogeneous. If the ANOVA was significant, group means were compared using the Dunnett test (2-sided).
If the Bartlett test was significant, means were compared using the non-parametric analysis of variance of Kruskal-Wallis. If the Kruskal-Wallis test was not significant (α = 0.05), the statistical procedure was stopped and group means were considered to be homogeneous. When the Kruskal-Wallis test was significant, group means were compared using the Dunn test (2-sided).
If one or more group variance(s) equaled 0, means were compared using non-parametric procedures. The levels of significance for each statistical comparison were 0.05 and 0.01.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 12000 ppm, in animals surviving to terminal sacrifice, treatment-related clinical signs consisted of white area on the eye in one male from Day 84 onwards and soiling around the anogenital region in one male on Day 36. In females, 5/10 animals had anogenital soiling on one or more occasion, 6/10 females had hair loss and 1 female had a wasted appearance on Day 90.
At 4000 ppm, clinical signs attributed to treatment consisted of white area on the eye in two males, on Days 71 to 84 in the first male and Days 50 to 90 in the second male. One male had piloerection on Day 90, but in the absence of other signs this isolated finding was considered to be incidental. One female had staining around the anogenital region on Day 90.
At 600 ppm, one male and one female had white area on the eye on Days 90 and 84, respectively. White area on the eye was also observed in one female on Day 90 at 1.25 ppm. In the absence of findings at the detailed ophthalmic examination, this finding was considered to be incidental and not treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male at 12000 ppm was found dead on Day 84. Clinical signs prior to death consisted of pale eyes, cold to touch, general pallor, reduced motor activity and red soiled fur. Body weight and food consumption were unaffected by treatment. A second male at 12000 ppm died during anesthesia for blood sampling on Day 86. No clinical signs were noted in-life for this animal, however, between Days 78 and 84 there was a 15 g loss in body weight with a corresponding reduction in food consumption during this period. At the macroscopic examination red soiling around the muzzle and a pale appearance were noted for this animal.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 12000 ppm, absolute body weight was significantly reduced from 9 to 12% in males and 6 to 10% in females throughout treatment. The effect on body weight gain was most pronounced after 1 week of treatment, where there was a 47% reduction in males and an 82% reduction in females, with an overall reduction at the end of 90 days of 19% in males and 13% in females. The effect was statistically significant in males after 1, 2 and 6 weeks of treatment and in females after 1 week of treatment.
At 4000 ppm, absolute body weight was reduced from 3 to 8% throughout the treatment period in females and was statistically significant at a number of time-points. Body weight gain in females was reduced by 47% after 1 week of treatment. The effect was statistically significant with an overall reduction of 10% after 90 days.
There was no effect on body weight in males at 4000 ppm or in either sex at 600 and 1.25 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Overall food consumption was reduced by 6% in males at 12000 ppm. The principal effect was observed at Week 1, where there was a 16% reduction compared to the controls. In females at 12000 ppm food consumption was reduced by 24% at Week 1. Thereafter food consumption was comparable to the controls.
At 4000 ppm, food consumption was reduced by 12% at Week 1 in females, but was similar to the control group thereafter.
Food consumption was comparable to the controls in males at 4000 ppm and in both sexes at 600 and 1.25 ppm.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

At 12000 ppm, one male had unilateral hemorrhaging in the iris at Week 3 together with unilateral/bilateral "snow flake" corneal opacity and neovascularisation of the cornea at Weeks 8 and 12. A second male at this dose level had unilateral hemorrhaging of the retina at Week 3 and bilateral pale retinal fundus at Week 12. No abnormalities were noted in females at this dose level.
At 4000 ppm, two males and one female had a unilateral "snow flake" corneal opacity and neovascularisation of the cornea. One male had these conditions at Weeks 3, 8 and 12, the second male at Weeks 8 and 12 and the female at Week 12 only. In addition, one female had unilateral hemorrhaging in the iris at Week 3.
At 600 ppm, one male and one female had a unilateral "snow flake" corneal opacity together with neovascularisation of the cornea at Week 12, in the male "snow flake" corneal opacity was also present at Week 8.
There were no findings at 1.25 ppm.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 12000 ppm in males, mean prothrombin time was higher (+13%) than the controls, though the effect was not statistically significant. One male which died under anesthesia for blood sampling had a severely low erythrocyte count and hemoglobin concentration, high leucocyte count with high neutrophil count and the presence of immature cells.
At 4000 ppm, a statistically significantly higher mean prothrombin time (+25%) was observed in males. One male had a moderately low erythrocyte count and hemoglobin concentration with a severely high mean corpuscular volume.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly higher mean total cholesterol concentrations were observed at 12000 ppm in both sexes (+45 and +34% in males and females, respectively) and at 4000 ppm and 600 ppm in males only (+47 and +43%, respectively). In males, this variation was not clearly dose-related.
Higher mean triglyceride concentrations were noted in males at 600, 4000 and 12000 ppm (+41, +46 and + 81%, respectively). However, only a few animals were affected in each group (5/10, 4/10 and 4/9 values out of the control range, respectively).
When compared to the control group, a slightly higher mean inorganic phosphorus concentration (+16%) was observed in females at 12000 ppm.
In the absence of relevant variation in the high dose group, the slightly higher mean total protein and albumin concentrations noted in males at 4000 and 600 ppm were considered to be of minor toxicological significance.
A tendency towards lower glucose concentrations (-25, -22 and -19%, respectively) were noted in females at 12000, 4000 and 600 ppm. However, as only a few animals were affected (2/10 values out of the control range in each group), these changes were considered not to be toxicologically relevant.
Some other statistically significant differences were noted, but were considered not to be toxicologically relevant in view of their occurrence and/or their low magnitude.
At 12000 ppm, the male which died under anesthesia had severely high transaminase activity, urea and triglycerides concentrations, severely low total protein concentration (both albumin and globulin) and moderately low sodium and calcium concentrations.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly higher urinary volume (+147%, p<0.01)) was seen in females at 12000 ppm. Tendencies towards lower pH values and towards higher ketone levels were noted at 600 ppm and above in both sexes.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean liver weights were statistically significantly higher in males at 600, 4000 and 12000 ppm. Mean liver to body weight ratio was found statistically significantly higher in females at 12000 ppm, but this change was associated with the lower mean terminal body weights and considered not to be toxicologically relevant.
Other statistically significant changes in kidneys and spleen were considered as incidental because they were not consistent and not associated to any other changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the male which was found dead on Day 84, the liver was found to be obviously enlarged, with multiple red foci and a prominent lobulation. The pancreas had multiple white and raised foci. Multiple red foci were observed within the thymus and dark contents within the intestines were observed. In addition, the adrenal gland was obviously enlarged.
The male which died on study Day 86 during anesthesia for blood sampling dark contents were found in the intestines. Multiple red foci were observed within the thymus and in the urinary bladder.
All other gross pathology changes observed were considered as incidental and not treatment-related.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of the two decedent animals at 12000 ppm revealed similar treatment related changes in the liver, pancreas and thymus. A diffuse mainly centrilobular acute hepatocellular necrosis was found in the liver. The lesion was marked in one and moderate in the second animal. A moderate acute/subacute pancreatitis with necrosis was observed within the pancreas. The thymus was characterized by moderate to marked single cell necrosis, mild to moderate atrophy/involution and slight multifocal parenchymal hemorrhages. Associated with these changes, multiple slight necrotic foci were found in several organs (adrenal gland, spleen, stomach, cerebellum) in one animal. A moderate atypical mixed cellular periarteritis with fibrosis was observed around the large vessels at the basis of the heart in the second animal. Additionally, another slight mixed cellular infiltrate was found focally within the serosa of the urinary bladder.
In animals sacrificed at study termination treatment-related changes were observed in the liver and the pancreas. In the liver, a slight to moderate diffuse centrilobular hepatocellular hypertrophy was observed in several males at 600, 4000 and 12000 ppm. This change was correlated to the higher liver weights. Additionally, pancreatic lesions were observed in four males at 12000 ppm. One male had a mild acute/subacute pancreatitis with necrosis, similar to the lesion observed in decedent animals. Four males had a slight to mild atypical periductular fibrosis and three males had interstitial edema. Focal interstitial intracytoplasmic golden brown pigment was present in two of them. No changes were observed in females at any dose level.
At 4000 ppm interstitial edema associated with an interstitial mixed cell infiltrate was observed in two males.
All other histological changes were considered to be incidental and not treatment-related, since they were consistent with changes commonly encountered in rats kept under laboratory conditions.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed at this dose level

Remarks on result:

other: equivalent to 259 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 787 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed at this dose level

Remarks on result:

other: equivalent to 902 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

12 000 ppm

System:

gastrointestinal tract

Organ:

pancreas

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Table 1. Mean body weights (g).

	Dose level (pm)
	0		1.25		600		4000		12000
	Male	Female	Male	Female	Male	Female	Male	Female	Male	Female
Day 90	541 ± 25	290 ± 17	533 ± 36	301 ± 27	514 ± 29	296 ± 15	514 ± 43	280 ± 21	479 ± 36**	273 ± 18

** (p ≤ 0.01) significantly different from controls

 Table 2. Ophthalmological findings during the treatmen period.

	Dose level (pm)
	600		4000		12000
	Male	Female	Male	Female	Male	Female
Cornea
Neovascularisation of the cornea	1/10	1/10	2/10	1/10	1/10	0/10
Corneal opacity "snow flake"	1/10	1/10	2/10	1/10	1/10	0/10
Iris
Hemorrhaging in the iris	0/10	0/10	0/10	1/10	1/10	0/10
Retina
Hemorrhaging in the retina	0/10	0/10	0/10	0/10	1/10	0/10
Fundus pale	0/10	0/10	0/10	0/10	1/10	0/10

 Table 3. Changes in clinical chemistry parameters.

	Dose level (pm)
	0		1.25		600		4000		12000
	Male	Female	Male	Female	Male	Female	Male	Female	Male	Female
Total cholesterol (mmol/L)	1.82 ± 0.30	2.00 ± 0.30	2.01 ± 0.31	2.24 ± 0.44	2.60 ± 0.37**	2.24 ± 0.46	2.67 ± 0.71**	2.39 ± 0.30	2.64 ± 0.39**	2.67 ± 0.38**
Triglycerides (mmol/L)	1.20 ± 0.27	0.57 ± 0.21	1.27 ± 0.52	0.62 ± 0.19	1.69 ± 0.61	0.59 ± 0.18	1.75 ± 0.67	0.78 ± 0.27	2.17 ± 1.07*	0.76 ± 0.14
Inorganic phosphorus	1.88 ± 0.17	1.49 ± 0.17	1.81 ± 0.14	1.51 ± 0.16	1.81 ± 0.09	1.48 ± 0.25	1.89 ± 0.18	1.58 ± 0.24	1.90 ± 0.18	1.73 ± 0.18*

* (p ≤ 0.05); ** (p ≤ 0.01) significantly different from controls

 Table 4. Liver weight changes.

	Dose level (pm)
	0		1.25		600		4000		12000
	Male	Female	Male	Female	Male	Female	Male	Female	Male	Female
Liver weight
Absolute (g)	11.5 ± 0.7	6.8 ± 0.8	12.0 ± 1.1	6.9 ± 0.6	14.4 ± 1.7**	6.9 ± 0.5	15.0 ± 2.1**	6.9 ± 1.1	14.0 ± 2.3**	7.0 ± 0.6
Relative to body weight (%)	2.23 ± 0.10	2.46 ± 0.32	2.38 ± 0.18	2.43 ± 0.27	2.93 ± 0.33**	2.48 ± 0.18	3.08 ± 0.32**	2.62 ± 0.29	3.08 ± 0.36**	2.76 ± 0.15*
Relative to brain weight (%)	527.84 ± 31.70	342.50 ± 37.68	562.13 ± 39.20	346.81 ± 36.28	693.03 ± 91.41**	352.48 ± 48	723.51 ± 73.31**	352.71 ± 48.29	692.52 ± 117.95**	346.05 ± 30.77

* (p ≤ 0.05); ** (p ≤ 0.01) significantly different from controls

 Table 5. Incidence and severity of treatment-related changes in the liver at terminal sacrifice.

	Dose level (pm)
	0		1.25		600		4000		12000
	Male	Female	Male	Female	Male	Female	Male	Female	Male	Female
Hepatocellular, hypertrophy, centrilobular, diffuse	0/10	0/10	0/10	0/10	4/10	0/10	10/10	0/10	6/10	0/10
Slight	-	-	-	-	4	-	7	-	4	-
Mild	-	-	-	-	-	-	2	-	2	-
Moderate	-	-	-	-	-	-	1	-	-	-

Conclusions:

The study was performed under GLP conditions and according to OECD 408. No Observed Adverse Effect Level (NOAEL) in the male Wistar rats when administered the test substance in the diet over a 90-day period was 4000 ppm (equivalent to 259 mg/kg/day) as in the 12000 ppm dose group two animals died and surviving animals showed histopathologic effects in the exocrine tissue of the pancreas. For female rats the NOAEL was set to 12000 ppm (equivalent to 902 mg/kg/day) since no adverse effects were observed at the highest dose level tested.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Oct 2002 - 22 Dec 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted in 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

no details on whether feed was analysed for contaminants

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Limit test:

no

Species:

rat

Strain:

other: Wistar Rj:WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: 180 - 252 g (males) and 133 - 186 g (females)
- Housing: 5 animals of same sex per cage in suspended, stainless steel and wire mesh cages with grided bottoms
- Diet: ground and irradiated rodent diet A04C-10P1 from SAFE (Scientific Animal Food and Engineering, Epinary-sur-Orge, France), ad libitum except at designated time periods
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: at least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 weeks
- Mixing appropriate amounts with (Type of food): A04C-10P1
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance (batch number R146) in the diet was demonstrated in a previous study (2002) at concentrations of 1.25 and 15000 ppm for at least 59 days under frozen storage followed by 11 days storage at room temperature. The stability of the test substance was also checked during the present study at concentrations of 2 and 5000 ppm for at least 77 days storage at room temperature.
The homogeneity of the test substance in the diet was verified for all first loads of the formulation F1, for the extreme concentrations on the first loads (2 and 5000 ppm) of the formulations F6, F13, F18 and F24, on the first load at 50 ppm of the formulation F6ter and on the first load at 2 ppm of the formulations F16bis and F17 to demonstrate adequate formulation procedures. Triplicate samples were collected of the lowest and the highest dose (900 and 18000 ppm) from the top, the middle and the bottom of the formulations. The results obtained from the homogeneity checks on the first loads were taken as measured concentration. Additionally, during the study, the concentration of all loads at each dietary level was verified (if not submitted for homogeneity check) prior to administration to the animals for the formulations F1, F2, F3, F6, F9, F13, F15, F16, F18, F21 and F24. The concentration of the preparations was also checked on the second loads of all supplemental preparations made for one week administration, on loads 2 to 4 at 50 ppm of the formulation F6ter, and on loads 2 to 4 at 2 ppm of the formulation F17.

Duration of treatment / exposure:

52 weeks (interim)
68 weeks (recovery group)
104 weeks (terminal sacrifice)

Frequency of treatment:

continuously (via diet)

Dose / conc.:

2 ppm

Remarks:

equivalent to approximately 0.09 and 0.13 mg/kg bw/day over the 12-month period in males and females, respectively and equivalent to approximately 0.08 and 0.11 mg/kg bw/day over the 24-month period in males and females, respectively

Dose / conc.:

50 ppm

Remarks:

equivalent to approximately 2.33 and 3.21 mg/kg bw/day over the 12-month period in males and females, respectively and equivalent to approximately 2.03 and 2.83 mg/kg bw/day over the 24-month period in males and females, respectively

Dose / conc.:

1 500 ppm

Remarks:

equivalent to approximately 72.0 and 99.6 mg/kg bw/day over the 12-month period in males and females, respectively and equivalent to approximately 62.4 and 88.6 mg/kg bw/day over the 24-month period in males and females, respectively

Dose / conc.:

5 000 ppm

Remarks:

equivalent to approximately 245 and 337 mg/kg bw/day over the 12-month period in males and females, respectively and equivalent to approximately 214 and 296 mg/kg bw/day over the 24-month period in males and females, respectively

No. of animals per sex per dose:

10 (12 month interim sacrifice)
15 (recovery group)
50 (24 month sacrifice)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results from a previous 90-day dietary study in the rat (2003).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily (including weekends and public holidays)
- Cage side observations included: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily (clinical signs), at least weekly (detailed physical examination including palpation for masses), except on Week 21 for females where physical examination was done twice for males in error

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 3, 7 and 10 during the acclimatisation period, weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied and of that remaining at the end of the food consumption period was recorded for each animal. Food consumption was recorded twice weekly during the first 6 weeks of treatment, then weekly up to Week 13, and once approximately every 4 weeks thereafter.The weekly mean achieved dosage intake in mg/kg bw/day for Weeks 1 to 13, then 1 week per month thereafter was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatization phase and at Month 4, 5, 12, 24 and at the end of reversibility phase
- Dose groups that were examined: all animals (acclimatization phase), all surviving animals (4, 5, 12, 24 months and after reversibility phase)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: all animals: Weeks 13 - 14, 22 - 24, 51 - 53; recovery group: on Week 69 (Day 91 of recovery phase); terminal scrifice: Weeks 78 and 105 - 107; puncture of the retro orbital venous plexus
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals (12 month interim sacrifice), first ten surviving animals (recovery group and 24 month terminal sacrifice)
- Parameters examined: Red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocyte count, white blood cell total and differential counts and platelet count, blood smears, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: all groups: Weeks 13 - 14, 22 - 24, 51 - 53; recovery group: on Week 69 (Day 91 of recovery phase); terminal sacrifice group: Weeks 78 and 105 - 107; puncture of the retro orbital venous plexus
- Animals fasted: Yes
- How many animals: all surviving animals (12 month interim sacrifice), first ten surviving animals (recovery group and 24 month terminal sacrifice)
- Parameters examined: Total bilirubin, glucose, urea, creatinine, total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyltransferase activities, total protein and albumin

URINALYSIS: Yes
- Time schedule for collection of urine: all groups: Weeks 11 - 12, 25 - 26 and 49 - 50; recovery groups: Week 68 (day 87 of recovery phase); terminal sacrifice group: Week 77 - 79 and 104
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: appearance, volume, pH, urinary refractive index, glucose, bilirubin, ketone bodies, occult blood, protein, urobilinogen, urinary sediment (microscopic exmination for grading of: red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
On Days 366, 367, 368 and 371 of the dosing phase for the interim sacrifice, on Days 94 and 97 of the recovery phase for the reversibility sacrifice and on Days 729 to 752 of the dosing phase for the terminal sacrifice, all surviving animals were sacrificed by exsanguination under deep anesthesia (pentobarbital, intraperitoneal injection of 60 mg/kg bw). An approximately equal number of animals randomly distributed amongst all groups were sampled on each day at the chronic and recovery sacrifice. At termination of the carcinogenicity phase, males were sacrificed prior to females due to the higher mortality rate in this sex. Consequently, an approximately equal number of males or females randomly distributed amongst the groups were sampled on each day taking into account the mortality. Animals were diet fasted overnight prior to sacrifices. All animals, including animals either found dead or killed for humane reasons, were necropsied. The necropsy included the examination of all major organs, tissues and body cavities. The organs or tissues checked in table 1 were sampled and/or weighed. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically. Samples were fixed by immersion in neutral buffered 10% formalin with the exception of the eye, optic nerve, Harderian gland, epididymis and testis that were fixed in Davidson's fixative. Two femoral bone marrow smears were prepared from sacrificed animals, one of which was stained with May-Grünwald Giemsa, but not examined as no relevant changes were observed in hematology or bone marrow histology. The second smear was stored unstained.


HISTOPATHOLOGY: Yes (see table 1)
Samples (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. Histological sections, stained with hematoxylin and eosin, were prepared from all organs and tissue samples. Histopathology examination of the chronic (52-week) phase was performed as follows:
- all organs and tissue samples from animals sacrificed or found dead during the treatment period,
- all organs and tissue samples from animals of control and high dose groups,
- liver, lung, kidneys, pancreas, eyes and gross abnormalities from animals of the intermediate dose groups.
Histopathological examinations were performed only on the liver, kidney, pancreas and eyes for animals which survived to scheduled sacrifice and all organs for animals which died before scheduled sacrifice. Histopathology examination of the carcinogenicity phase was performed on all organs and tissues embedded including gross abnormalities in all animals from all groups including decedents. For all unscheduled sacrificed or dead animals on study, the cause of death (whether or not a tumor was the cause of death) was determined when it was possible.

Statistics:

In general Bartlett test was performed. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p>0.05), the group means were considered homogeneous and no further analysis was performed. If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p>0.05), group means were considered homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for body weight and average food consumption/day parameters and selected haematology parameters (red blood cell count, platelet count, white blood cell count, neutrophil count, lymphocyte count, reticulocyte count) data were transformed using the log transformation for body weight and food consumption and the square root transformation for haematology parameters before the same methods were applied as mentioned above. If the Bartlett test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded as mentioned above.
Individual statistic methods used for specific parameters are descibed in `Any other information on materials and methods´.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES
50 ppm: white area on eyes (45/60)
1500 ppm: white area on eyes (49/60)
5000 ppm: cold appearance (7/75), dental abnormality (13/75), general pallor (9/75), reduced motor activity (9/75), soiled anogenital region (7/75), localized soiled fur (7/75), wasted appearance (5/75) and white area on eyes (53/75; progressively disappearing within 3 month recovery, except of 1 animal)

FEMALES
2 ppm: white area on eyes (13/75)
50 ppm: white area on eyes (48/60)
1500 ppm: white area on eyes (55/60), soiled anogenital region (4/60)
5000 ppm: soiled anogenital region (12/75), white area on eyes (58/75; progressively disappearing within 3 month recovery) and hair loss (23/75)

Second year:
MALES
50 ppm: focal swelling (10/48; principally of hindlimb), reduced motor activity (12/48) and white area on eyes (41/48)
1500 ppm: focal swelling (21/45; principally of hindlimb), white area on eyes (45/45) and reduced motor activity (9/45; not statistically significant)
5000 ppm: focal swelling (16/42; principally of hindlimb), reduced motor activity (11/42), skin lesions (13/42) and white area on eyes (41/42)

FEMALES
50 ppm: white area on eyes (42/50)
1500 ppm: white area on eyes (45/49)
5000 ppm: white area on eyes (38/49), chromodacryorrhea (10/49) and soiled anogenital region (6/49)

For more details see table 2 in section "any other information on results incl. tables".

Mortality:

mortality observed, treatment-related

Description (incidence):

MALES
0 ppm: 1/75 animals after 1 year, 29/50 animals after 2 years
2 ppm: 4/60 animals after 1 year, 33/50 animals after 2 years
50 ppm: 2/60 animals after 1 year, 40/50 animals after 2 years
1500 ppm: 5/60 animals after 1 year, 34/50 animals after 2 years
5000 ppm: 16/75 animals after 1 year, 33/50 animals after 2 years

Only death of high dose group males where 16/75 were found dead or were sacrificed prematurely for humane reasons, were considered to be treatment-related. However, this effect was transient, as the overall mortality rate over two years was similar to the controls.

FEMALES
0 ppm: 0/75 animals after 1 year, 23/50 animals after 2 years
2 ppm: 1/60 animals after 1 year, 27/50 animals after 2 years
50 ppm: 0/60 animals after 1 year, 14/50 animals after 2 years
1500 ppm: 2/60 animals after 1 year, 17/50 animals after 2 years
5000 ppm: 1/75 animals after 1 year, 13/50 animals after 2 years

For more details see table 3 in section "any other information on results incl. tables".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES
2 and 50 and ppm: Mean body weight and body weight gain were comparable to the controls.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 6 and 11% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control during the first week of treatment, mean body weight and mean body weight gain were lower by 5 and 21% (p<0.01), respectively. Compared to the control group by the end of first year, mean body weight and mean body weight gain were lower by 4 and 6%(p<0.05), respectively.

FEMALES:
2 and 50 and ppm: Mean body weight and body weight gain were comparable to the controls.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 6 and 18% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control group during the first week of treatment, mean body weight and mean body weight gain were lower by 4 and 29% (p<0.01), respectively. Compared to the control group by the end of first year, mean body weight and mean body weight gain were lower by 6 and 11% (p<0.01), respectively.

Recovery period: During the 3-month recovery period, mean weekly body weight gain in both sexes previously treated at 5000 ppm was comparable to the controls or slightly reduced.

Second year:
MALES
2 ppm: Mean body weight and body weight gain were comparable to the controls.
50 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 11 and 17% (p<0.01 or p<0.05), respectively.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 12 and 17% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control mean body weight and mean body weight gain were lower by 12 and 17% (p<0.01 or p<0.05), respectively.

FEMALES:
2 ppm: Mean body weight and body weight gain were comparable to the controls.
50 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 8 and 12% (p<0.01 or p<0.05), respectively.
1500 ppm: Compared to control mean body weight and mean body weight gain were slightly lower by up to 16 and 23% (p<0.01 or p<0.05), respectively.
5000 ppm: Compared to the control mean body weight and mean body weight gain were lower by 16 and 23% (p<0.01 or p<0.05), respectively.

For more details see tables 4 and 5 in section "any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

First year:
MALES
5000 ppm: decreased mean food consumption (6%; not statistically significant) in first weeks of treatment

FEMALES
5000 ppm: decreased mean food consumption (5%; p<0.05) in first weeks of treatment

The other few minor differences (reaching statistical significance) from controls were considered to reflect interindividual variation rather than a treatment-related effect.

Recovery phase:
During the 3-month recovery period, no relevant change was observed in the animals previously treated at 5000 ppm during one year.

Second year:
Mean food consumption was similar to controls during the second year of the study in both sexes and at all dose levels. The few minor increases (reaching statistical significance) in males at 5000 ppm were considered to reflect inter-individual variation rather than a treatment-related effect.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

First year:
At 5000, 1500 and 50 ppm, the following treatment-related ophthalmological findings were observed after 4 months of exposure: corneal opacity, neovascularization of the cornea and snow flake-like corneal opacity in both sexes, in association with oedema of the cornea in males only. The incidence of these findings was already marked by Month 4 and increased throughout the duration of the study. The type of corneal lesions observed at 5000, 1500 and 50 ppm in the rat (together with the eye keratitis noted at the microscopic examination), are based on characteristics of the compound that inhibits 4-hydroxyphenylpyruvic acid
dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. These lesions are related to an increase in plasma tyrosine level caused by a blockade of the 4-HPPDase enzyme in the rat. However, the rat is a species particularly sensitive to inhibition of the 4-HPPDase enzyme and is atypical in its susceptibility to develop tyrosine-related eye lesions. Therefore, although these lesions (corneal lesions and eye keratitis) were treatment-related, they were considered not to be toxicologically relevant to man. At 2 ppm, no treatment-related ophthalmological change was noted, as the incidences of ophthalmological findings were similar to the control groups at all time points.Other ophthalmological findings recorded were those commonly recorded or were recorded in one
or two animals only and were thus considered not to be related to test substance administration.

Recovery phase:
After 13 weeks of recovery, corneal opacity, oedema of the cornea and snow flake-like corneal opacity were totally reversible whilst neovascularization of the cornea persisted in some high dose group animals affected during the chronic phase.

Second year:
At the end of 2 years of exposure, the following treatment-related ophthalmological findings were observed:
At 5000 ppm, the incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity was statistically significantly elevated in both sexes. At 1500 and 50 ppm, the incidence of corneal opacity, neovascularization and oedema of the cornea was statistically significantly elevated in both sexes, in association with a statistically significant increase in the incidence of snow flake-like corneal opacity in females. At 2 ppm, ophthalmological examination did not reveal any statistically significant changes. Other ophthalmological findings recorded were those commonly recorded or were recorded in one or two animals only and were thus considered not to be related to the test substance administration.

For more details see table 6 in section "any other information on results incl. tables".

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES: Increased total cholesterol levels (up to 57%; p<0.01) and/or triglyceride levels (up to 72%; p<0.01) in males and femles, respectively, at concentrations of 50, 1500 and 5000 ppm; decreased mean alkaline phosphatase activity (up to -27%; p<0.05) at 5000 and 1500 ppm at Months 3 and 6; slight variations (not toxicologically relevant) in total protein (up to 13%; p<0.01) and albumin concentrations (up to 12%; p<0.01) and consequently in globulin concentrations and albumin/globulin ratio (calculated parameters) at 5000, 1500 and 50 ppm at Month 3

FEMALES: Increased mean total cholesterol (up to 32%; p<0.01) and/or triglycerides concentrations (up to 20%; p<0.01) at 50, 1500 and 5000 ppm

Recovery period: All changes detected during first year were found to be reversible, as after the 3-month recovery period.

Second year:
MALES: Increased mean total cholesterol concentrations (+36%; p<0.05) at 5000 ppm after 18 month but reversible within 24 month

FEMALES: Increased mean total cholesterol concentrations (+27%; p<0.05) at 5000 ppm after 18 month but reversible within 24 month

For more details see table 7 in section "any other information on results incl. tables".

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES: Higher ketone level at Month 3 (5000 and 1500 ppm), Month 6 (5000 ppm) and Month 12 (5000, 1500 and 50 ppm); lower mean pH (50, 1500 and 5000 ppm); higher protein level Month 3 (5000 ppm) and at Months 6 and 12 (5000, 1500 and 50 ppm); lower amount of crystals (50, 1500 and 5000 ppm)

FEMALES: Higher ketone level at all sampling periods at 50, 1500 and 5000 ppm; lower mean pH (50, 1500 and 5000 ppm)

Recovery period: All changes detected during the chronic phase were found to be reversible after the 3-month recovery period, with the exception of the mean pH value that remained lower in the females previously treated at 5000 ppm for one year.

Second year:
MALES: Higher ketone level at all sampling periods at 50, 1500 and 5000 ppm; lower mean pH values at Months 18 and 24 (50, 1500 and 5000 ppm); higher protein levels and lower amounts of crystals at Month 18 only (50, 1500 and 5000 ppm)

FEMALES: Higher ketone level at all sampling periods at 50, 1500 and 5000 ppm

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

First year:
MALES: higher mean absolute and/or relative liver and kidney weights at 50, 1500 and 5000 ppm compared to control; increased brain weight by 7 and 6% at 1500 and 5000 ppm, respectively

Recovery phase:
Mean terminal body weight was lower in both sexes at 5000 ppm (-10 and -12% in males and females, respectively, p<0.05), when compared to controls. All organ weight changes detected at the end of the chronic (first year) phase were found to be reversible and therefore not toxicologically relevant. The few changes noted after the 3-month recovery period were considered to be incidental in view of their individual variation.

Second year:
MALES: higher mean abosolute and/or relative liver and kidney weights (statistically significant for most parameters) at 50,1500 and 5000 ppm compared to control; decreased mean absolute brain weight at 50, 1500 and 5000 ppm (not toxicological relevantas since they were observed for one parameter only, were opposite in both sexes and were not associated with any change at the macroscopic and microscopic examinations)

FEMALES: higher mean abosolute and relative liver weights at 1500 and 5000 ppm compared to control; lower absolute and mean kidney to brain weights at 1500 ppm (non-treatment related since no dose-relationship); decresed ovary weights at 5000 ppm (this increase was attributable to a bilateral mass noted on ovary of this female, which contributed by itself to the increase in ovary weights in this group) and 50 and 1500 ppm (not toxicological relevant since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations); increased brain to body weight at 1500 and 5000 ppm (not toxicological relevantas since they were observed for one parameter only, were opposite in both sexes and were not associated with any change at the macroscopic and microscopic examinations)

For more details see table 8 in section "any other information on results incl. tables".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

First year:
- Unscheduled death:
MALES
5000 ppm: haemorrhagic sydrome in 4 animals found dead at the beginning of the study: multiple heamorrages in the subcutis, brain and testis in 2 animals found dead on Day 9 and 25; subcutaneous haematoma in 2 animals found dead on Day 24 and 25; The haemorrhagic lesions were the cause of death and were considered to be treatment-related in these 4 animals; non treatment-related findings: irregular surface of the kidney and bilateral eye opacity in one animal killed on Day 24

- Terminal sacrifice:
MALE/FEMALE: eye opacity at 50, 1500 and 5000 ppm

Recovery period:
- Unschedules death:
MALES: dark contents in the stomach and intestines in two animals found dead on Day 29, red foci in the epidymidis, brain haematoma, uniliteral eye opacity in one animal found dead on Day 11
FEMALES: irregular surface on the kidney in one animal found dead on Day 55

Second year:
- Unscheduled death:
MALES:
50 ppm: eye opacity
1500 ppm: eye opacity
5000 ppm: eye opacity, pale appearance of animals

FEMALES
1500 ppm: eye opacity
5000 ppm: irregular surface of kidney, skin allopecia (not toxicological relevant since no relevant microscopic changes occured)

- Terminal sacrifice:
MALES
50 ppm: eye opacity, irregular surface on kidney
1500 ppm: eye opacity, irregular surface on kidney (not statistically significant)
5000 ppm: eye opacity, irregular surface on kidney

FEMALES
50 ppm: eye opacity, enlarged pituitary gland (not toxocilogically relevant since no dose-relationsship)
1500 ppm: eye opacity
5000 ppm: eye opacity, enlarged pituitary gland (not toxocilogically relevant since no dose-relationsship)

For more details see tables 9 and 10 in section "any other information on results incl. tables".

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

First year:
- Unscheduled death: haemorrhagic syndrome in 4/9 animals found dead; progressive nephropathy and keratitis in one male killed on Day 241
- Terminal sacrifice:
MALES:
Kidney: chronic progressive nephropathy at 50, 1500 and 5000 ppm (including: tubular cell regeneration, thickened basement membranes (glomerular and tubular), interstitial fibrosis, inflammation, dilated/cystic tubules, protein casts, debris, mesangial proliferation, glomerular sclerosis, and hypertrophy/hyperplasia of tubular epithelium)

Pancreas: minimal to moderate acinar atrophy/fibrosis (focal/multifocal) at all dose levels (non-treatment-related since no dose-relationship)

Eye: minimal to marked keratitis (including following changes in the cornea: acute to chronic active inflammation, keratinization, epithelial vacuolization, neovascularization) at 50, 1500 and 5000 ppm (not relevant for human since inhibition of key enzyme provoking systemic tyrosinaemia is relevant in the rat only)

Liver: centrilobular hepatocellular hypertrophy (diffuse) in 2/10 animals at 1500 ppm and in 3/5 animals at 5000 ppm (adaptive response)

FEMALES:
Pancreas: minimal to moderate interstitial inflammation (focal/multifocal) at 5000 ppm (not toxicological relevant since no dose-effect); minimal to moderate acinar atrophy/fibrosis (focal/multifocal) at all dose levels (non-treatment-related since no dose-relationship)

Eye: minimal to marked keratitis (including: acute to chronic active inflammation, keratinization, epithelial vacuolization, neovascularization) at 50, 1500 and 5000 ppm (not relevant for human since inhibition of key enzyme provoking systemic tyrosinaemia is relevant in the rat only)

Urinary bladder: transitional cell hyperplasia in 5/10 animals at 5000 ppm (reversible since not observed after 24 months)
Adrenal gland: zona glomerulosa hypertrophy in 7/10 animals at 5000 ppm (not toxicological relevant since low severity of findings and reversible since not observed after 24 months)

Recovery period:
- Unscheduled death: hemorrhagic syndrome in 3 males found dead at beginning of recovery period; Kidney: progressive nephropathy in one female found dead on Day 55

- Terminal sacrifice:
MALES
5000 ppm:
Kidney: minimal to severe chronic progressive nephropathy and minimal to slight interstitial inflammation (focal/multifocal)
Eye: keratitis (not relvant for human; see comment at chronic phase)

FEMALES
5000 ppm:
Pancreas: interstitial inflammation iin 7/14 animals; minimal to slight acinar atrophy/fibrosis (non-treatment-related); basophilic changes in 4/14 animals (non-treatment related)
Eye: keratitis (not relvant for human; see comment at chronic phase)

Second year:

MALES:
Kidney: minimal to severe chronic progressive nephropathy at 50, 1500 and 5000 ppm (no relevance for human since age related, spontaneously occuring in rats and a high incidence in control gorup)

Pancreas: minimal to moderate interstitial inflammation (focal/multifocal) in the pancreas at 1500 and 5000 ppm (not toxicologically relevant since no real difference in severity to controls); minimal to severe acinar atrophy/fibrosis (focal/multifocal) at 1500 and 5000 ppm (statistically significant)

Thyroid gland: colloid alteration and brown pigment in the cytoplasm of the thyroid follicular cells (not toxicologically relevant since similar alterations are present in control rats, indicating that these changes likely reflect a normal physiologic process associated with the unique rapid turnover of colloid) and follicular cell hypertrophy at 50, 1500 and 5000 ppm

Eye: marked keratitis at 50, 1500 and 5000 ppm (not toxicological relevant to human, see comment at chronic phase); atrophy/degeneration (multifocal/diffuse) in sciatic nerve at 50, 1500 and 5000 ppm (non-treatment related since high incidence in control groups and no dose-dependency)

Skeletal muscle: minimal to marked myofiber atrophy/degeneration at 50, 1500 and 5000 ppm (non-treatment related since secondary effect from sciatic nerve atrophy),

Hindleg/hindpawn: ulcerative necrotising inflammation at 1500 and 5000 ppm (indirect effect with minor toxicological relevance)

Liver: angiectasis (focal/multifocal) and extramedullary hematopoiesis (multifocal) at 1500 and 5000 ppm (not toxicologically relevant since age related and no dose-dependency); minimal to slight centrilobular hepatocellular hypertrophy (diffuse) at 1500 ppm (adaptive response)

Brain: minimal to slight mineralization (focal/multifocal) at 5000 ppm (non-treatment-related since also observed in control groups and within range of historical control data)

Pituitary gland: cyst(s)/pseudocyst(s) in pars distalis at 5000 ppm (not toxicologically relevant since it was a spontaneous finding)

Urinary bladder: proteinaceous material at 5000 ppm of animals found dead (not toxicologically relevant since it is a common finding and was obtained in isolation for this parameter)

FEMALES:
Pancreas: minimal to moderate interstitial inflammation (focal/multifocal) at 50, 1500 (not significant) and 5000 ppm (not toxicologically relevant since no real difference in severity to controls); minimal to severe acinar atrophy/fibrosis (focal/multifocal) at 2 (within range of historical data), 50, 1500 and 5000 ppm

Thyroid gland: colloid alteration, brown pigment in the cytoplasm of the thyroid follicular cells (not toxicologically relevant since similar alterations are present in control rats, indicating that these changes likely reflect a normal physiologic process associated with the unique rapid turnover of colloid) and follicular cell hypertrophy at 50, 1500 and 5000 ppm

Eye: marked keratitis at 50, 1500 and 5000 ppm (not toxicological relevant to human, see comment at chronic pahse); atrophy/degeneration (multifocal/diffuse) in sciatic nerve at 1500 ppm (non-treatment related since high incidence in control groups and no dose-dependency)

Skeletal muscle: minimal to marked myofiber atrophy/degeneration at 1500 and 5000 ppm (non-treatment related since secondary effect from sciatic nerve atrophy)

Liver: bile duct hyperplasia (focal/multifocal) at 50, 1500 and 5000 ppm (not toxicologically relevant since age related and no dose-dependency)

Ovaries: minimal to marked diffuse atrophy at 5000 ppm (non-treatment-related since within the range of historical control data)

For more details see table 11 in section "any other information on results incl. tables".

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Remarks:

24 month

Effect level:

2 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Remarks on result:

other: equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

LOAEL

Remarks:

24 month

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 2.03 and 2.83 mg/kg bw/day in males and females, respectively

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm

System:

gastrointestinal tract

Organ:

pancreas

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 2. Treatment-related clinical signs

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
First year (chronic phase)
Number of animals examined	75	60	60	60	75	75	60	60	60	75
Cold to touch	0	0	0	0	7*	nc	nc	nc	nc	nc
Dental abnormality	5	6	6	2	13*	nc	nc	nc	nc	nc
General pallor	0	0	0	1	9**	nc	nc	nc	nc	nc
Reduced moto activity	0	0	0	0	9**	nc	nc	nc	nc	nc
Soiled anogenital region	0	0	0	1	7**	0	0	2	4*	12**
Soiled fur, localized	0	0	1	1	7**	nc	nc	nc	nc	nc
Wasted	0	1	0	1	5*	nc	nc	nc	nc	nc
White area in eyes	1	1	45**	49**	53**	2	13**	48**	55**	58**
Hair loss	nc	nc	nc	nc	nc
Recovery phase
Number of animals examined on Week 1 of recovery	15	-	-	-	12	14	-	-	-	15
White area on eyes	0	-	-	-	9**	0	-	-	-	9**
Number of animals examined on Week 13 of recovery	15	-	-	-	11	14	-	-	-	14
White area on eyes	0	-	-	-	1	0	-	-	-	1
Second year (carcinogenicity phase)
Number of animals examined	21	17	10	16	17	27	22	36	33	36
Focal swelling	3	3	10*	21**	16**	nc	nc	nc	nc	nc
Reduced motor activity	5	6	12*	9	11*	nc	nc	nc	nc	nc
Skin lesions	7	10	6	9	13*	nc	nc	nc	nc	nc
White area on eye	3	3	41**	45**	41**	2	6	42**	45**	38**
Chromodacryorrhea	nc	nc	nc	nc	nc	1	1	3	4	10**
Soiled anogenital region	nc	nc	nc	nc	nc	0	0	0	3	6*

*•: p<0.05; **: p<0.01; nc: not concerned or no treatment-related change.

Table 3. Mortality incidence after 1 and 2 years of treatment (unscheduled deaths of animals)

	First year of treatment (chronic phase)
Dose level (ppm)	0	2	50	1500	5000
Males
Number of animals	75	60	60	60	75
Mortality	1	4	2	5	16
Females
Number of animals	75	60	60	60	75
Mortality	0	1	0	2	1
	Second year of treatment (carcinogenicity phase)
Males
Number of animals	50	50	50	50	50
Mortality	29	33	40	34	33
Females
Number of animals	50	50	50	50	50
Mortality	23	27	14	17	13

 Table 4. Body weights and body weight gains in males

Dose level [ppm]	0	2		50			1500		5000
	g	g	% of control		g	% of control	g	% of control	g	% of control
BODY WEIGHTS
Initial (Day 1)	218	218	100		217	100	216	99	215	99
Week 1 (Day 8)	276	278	101		278	101	270	98	261**	95
Week 13 (Day 92)	535	547	102		538	101	504**	94	500**	93
Week 25 (Day 176)	630	639	101		631	100	602*	96	585**	93
Week 49 (Day 344)	708	709	100		706	100	682	96	677*	96
Week 77(Day 372)	737	742	101		685*	93	664**	90	671**	91
Final (Day 708)	660	679	103		606	92	600*	91	630	95
BODY WEIGHT GAINS
Week 1 (Days 1 – 8)	58	61	105		61	105	54**	93	46**	79
Weeks 1 – 13 (Days 1 – 92)	318	329	103		320	101	288**	91	284**	89
Weeks 13 – 25 (Days 92 – 176)	95	91	96		94	99	96	101	86	91
Weeks 25 - 49 (Days 176 – 344)	79	73	92		75	95	81	103	92	116
Weeks 49 – 77 (Days 344 – 540)	38	36	95		-17**	nc	-11	nc	-11**	nc
Weeks 77 – 101 (Days 540 – 708)	-47	-54	115		-64	136	-67	143	-44	94
Overall (Days 1 – 708)	446	463	104		392	88	383	86	418	94

*: p≤0.05; **: p≤0.01; nc: not calculated

 Table 5. Body weights and body weight gains in females

Dose level [ppm]	0	2		50			1500		5000
	g	g	% of control		g	% of control	g	% of control	g	% of control
BODY WEIGHTS
Initial (Day 1)	162	162	100		162	100	162	100	163	101
Week 1 (Day 8)	190	190	100		189	99	185*	97	182**	96
Week 13 (Day 92)	291	291	100		289	99	280**	96	282*	97
Week 25 (Day 176)	327	324	99		325	99	317*	97	318*	97
Week 49 (Day 344)	363	356	98		359	99	345*	95	341**	94
Week 77(Day 372)	433	428	99		412	99	385**	89	393**	91
Final (Day 708)	474	483	102		438	92	405**	85	401**	85
BODY WEIGHT GAINS
Week 1 (Days 1 – 8)	28	28	100		26	93	23**	82	20**	71
Weeks 1 – 13 (Days 1 – 92)	129	128	99		127	98	118**	91	119**	92
Weeks 13 – 25 (Days 92 – 176)	37	33	89		36	97	37	100	36	97
Weeks 25 - 49 (Days 176 – 344)	36	33	92		34	94	27	75	23*	64
Weeks 49 – 77 (Days 344 – 540)	71	69	97		52	73	43**	61	45**	63
Weeks 77 – 101 (Days 540 – 708)	42	52	124		32	76	28	67	13**	31
Overall (Days 1 – 708)	309	321	104		276	89	245**	79	239**	77

*: p≤0.05; **: p≤0.01; nc: not calculated

Table 6. Ophthalmological findings

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Month 4 (Weeks 14 - 15)
Number of animals examined	75	60	60	59	63	75	60	60	59	75
Corneal opacity	0	0	12**	14**	8**	0	0	5*	7**	4
Neovascularization of the cornea	0	0	30**	38**	34**	1	1	30**	47**	23**
Oedema of the cornea	0	0	12**	12**	6**	1	2	1	1	2
Corneal opacity « snow flake »	0	0	22**	27**	29**	0	0	29**	41**	22**
Month 5 (Weeks 21 – 22)
Number of animals examined	75	60	59	59	63	75	60	60	59	75
Corneal opacity	0	0	23**	21**	22**	0	0	7**	25**	9**
Neovascularization of the cornea	0	0	33**	40**	39**	1	0	40**	50**	37**
Oedema of the cornea	0	0	23**	22**	22**	1	1	4	24**	9**
Corneal opacity « snow flake »	0	0	13**	22**	18**	0	0	35**	28**	32**
Month 12 (Weeks 50 – 51)
Number of animals examined	75	60	59	59	63	75	59	60	58	74
Corneal opacity	0	0	36**	37**	36**	1	1	21**	41**	28**
Neovascularization of the cornea	0	0	44**	45**	49**	1	1	46**	53**	50**
Oedema of the cornea	0	0	36**	39**	45**	1	2	26**	45**	37**
Corneal opacity « snow flake »	0	0	8**	8**	17**	0	0	27**	16**	24**
Week 13 of recovery phase
Number of animals examined	15	-	-	-	11	14	-	-	-	14
Neovascularization of the cornea	0	-	-	-	2	0	-	-	-	6**
Month 24 – 25 (Weeks 104 – 106)
Number of animals examined	21	17	11	17	17	29	25	37	33	39
Corneal opacity	0	0	10**	16**	11**	0	3	15**	26**	19**
Neovascularization of the cornea	0	0	10**	17**	17**	0	3	30**	32**	30**
Oedema of the cornea	0	0	10**	17**	17**	0	3	20**	31**	25**
Corneal opacity « snow flake »	0	0	0	1	7**	0	0	16**	12**	12**

*: p≤0.05; **: p≤0.01

Table 7. Changes in clinical chemistry parameters compared to controls [%]

Dose level (ppm)	50			1500			5000
Month	3	6	12	3	6	12	3	6	12
Cholesterol concentration
Males	+20%*	+20%	+38%**	+21% *	+24%**	+29**	+46%**	+44%**	+57%**
Females	-4%	+9%	-1%	+6%	+13%*	+12%	+24%**	+26%**	+32%**
Triglyceride concentration
Males	+44%*	+15%	+42%*	+48%*	+50%	+44%	+51%**	+34%*	+72%**
Females	+40%**	+9%	+11%	+49%**	+21%*	+6%	+62%**	+18%	+20%
Alkaline phosphatase activity
Males	-23%	-11%	-2%	-27%*	-23%**	-20%	-32%**	-20%**	-17%

*: p≤0.05; **: p≤0.01

Table 8. Changes in Organ weights

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
First year (chronic phase)
Number of animals examined	10	9	9	10	5	10	10	10	8	10
Mean absolute liver weight (g)	13.8	13.0	16.5	16.3	16.7	7.5	7.9	8.0	8.5	7.8
Mean liver to bw ratio (%)	2.00	1.92	2.38**	2.44**	2.67**	2.30	2.33	2.34	2.56	2.45
Mean absolute kidney weight (g)	3.45	3.47	4.04*	3.94	3.63	2.32	2.47	2.39	2.36	2.26
Mean kidney to bw ratio (%)	0.501	0.510	0.586*	0.593**	0.589*	0.710	0.730	0.695	0.709	0.715
Second year (carcinogenicity phase)
Number of animals examined	21	17	10	16	17	27	22	36	33	36
Mean absolute liver weight (g)	12.6	13.5	14.0	15.7**	15.7**	10.5	11.0	10.6	10.1	10.7
Mean liver to bw ratio (%)	2.09	2.09	2.51**	2.84**	2.74**	2.40	2.52	2.63	2.69*	2.81**
Mean absolute kidney weight (g)	0.665	0.673	0.801	0.921**	0.812*	0.709	0.706	0.725	0.736	0.766
Mean kidney to bw ratio (%)	168.4	182.4	196.7*	225.7**	208.3**	143.1	139.2	137.1	128.8**	137.9

*: p≤0.05; **: p≤0.01

Table 9. Macroscopic changes at terminal sacrifice of first year and recovery phase

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
First year (chronic phase)
Eye opacity	0/10	0/9	6/9**	4/10*	2/5	1/10	0/10	6/10*	4/8	7/10**
Second year (carcinogenicity phase)
Eye opacity	1/21	1/17	7/10**	9/16**	14/17**	1/27	3/23	22/36**	30/33**	23/37**
Irregular surface on kidney	2/21	2/17	7/10**	6/16	7/17*	nc	nc	nc	nc	nc

*: p≤0.05; **: p≤0.01

Table 10. Macroscopic changes at unscheduled sacrifice (incidence) in second year (carcinogenicity phase)

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
Eye opacity	1/29	3/33	10/40*	13/34**	11/33**	1/23	1/27	1/14	6/17	3/13
Irregular surface on kidney	nc	nc	nc	nc	nc	0/23	0/27	1/14	1/17	3/13*

*: p≤0.05; **: p≤0.01

 Table 11. Microscopic changes at terminal sacrifice

	Males					Females
Dose level (ppm)	0	2	50	1500	5000	0	2	50	1500	5000
First year (chronic phase)
Bilateral keratitis	0/10	0/9	6/9**	2/10	1/5	0/10	0/10	8/10**	6/8**	5/10*
Unilateral keratitis	0/10	0/9	3/9	3/10	2/5	0/10	0/10	1/10	1/8	5/10*
Recovery phase
Chronic progressive nephropathy	5/15	-	-	-	9/11	0/14	-	-	-	2/14
Interstitial inflammation of pancreas	2/15	-	-	-	7/11	1/14	-	-	-	3/14
Unilateral or bilateral keratitis in the eye	1/15	-	-	-	6/11	0/14	-	-	-	7/14
Second year (carcinogenicity phase)
Follicular cell hypertrophy in thyroid gland	3/50	6/47	16/48**	19/50**	27/48**	1/50	3/50	22/50**	20/49**	19/50**
Chronic progressive nephropathy	30/50	35/50	43/50**	42/50**	38/50**	16/50	17/50	25/50	20/50	24/50
Acinar atrophy/fibrosis in the pancreas	18/50	24/48	23/49	33/49**	36/48**	13/50	22/50*	28/50**	32/48**	31/50**
Unilateral or bilateral keratitis in the eye	1/49	1/50	41/49**	47/49**	42/50**	1/50	2/50	36/50**	44/49**	38/50**
Ulcerative necrotising inflammation in hindleg and/or hindpawn of samples	3/7	3/7	7/13	13/13	18/21	1/1	0/0	3/3	3/5	2/4

*: p≤0.05; **: p≤0.01

Conclusions:

The study was performed under GLP conditions and according to OECD 453. The NOAEL over a 24-month period of dietary administration with the test substance for males and females was 2 ppm for both (equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively). Based on this data, the test substance meets the criteria for classification according to Regulation (EC) No 1272/2008, and has therefore to be classified as STOT RE Cat. 1 with pancreas (exocrine) and thyroid gland as target organs.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jun 2005 - 06 Nov 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Detailed results tables are missing in the translated version of the study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

yes

Remarks:

detailed results tables and historical control data are missing in the translated study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

detailed results tables and historical control data are missing in the translated study report

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KITAYAMA LABES Co., Ltd (Hongo farm, Japan)
- Age at study initiation: 7 months
- Weight at study initiation: 8.3 - 10.4 kg (males), 8.5 - 10.5 kg (females)
- Housing: Animals were housed individually in stainless steal cages (width 835 mm × depth 900 mm × height 800 mm) sustained in racks equipped with automatic sweepers
- Diet: Certified pellet diet DS-A (Oriental Yeast Co. Ltd., Tokyo, Japan)
- Water: Well water (ad libitum), pressed through a rapid filtration unit with sand filter and an adsorption unit with charcoal filter, was sterilized with sodium hypochlorite and ultraviolet light
- Acclimation period: 54 days (males), 62 days (females)

DETAILS OF FOOD AND WATER QUALITY:
- Food: The supplier analysed each lot of the basal diet for the following nutrients - moisture, crude protein, crude fat, crude ash, crude fibre and nitrogen-free extract
- Food: The supplier analysed each lot of the basal diet for the following contaminants - mercury, cadmium, lead, chromium, arsenic, selenium, γ-BHC, DDT (and related compounds), adrin, dieldrin, endrin, heptachlor, malathion, parathion, aflatoxin (B1, B2, G1 and G2), polychlorobiphenyl, estradiol, dimethylnitrosamine, diethylnitrosamine
- Water: Analysed for arsenic, lead, total mercury, cadmium, selenium, polychlorinated biphenyl, n-nitroso compounds, total trihalomethanes, simazine, thiram, thiobencarb, 1,3-dichloropropene

Analysis confirmed contaminant level were within the acceptable ranges for food and wate.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): more than 10 times per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Details on route of administration:

The test substance was incorporated into the basal diet and administered to animals of both sexes continuously for a period of 52 weeks.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly for the first 16 weeks, every 4 weeks thereafter.
- Mixing appropriate amounts with (Type of food): The test substance was mixed with part of the basal diet in a pestel and mortar, then this premix was mixed with the rest of the basal diet by means of a blending machine, HP-60 or SS-501.
- Storage temperature of food: Cold, dark and sealed conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test substance in diet when stored at room temperature for 8-days or under cold, dark and sealed conditions for 5 weeks had been demonstrated in a previous study at 100 and 2000 ppm (Study no. IET 04-5010, 2007). However, as the current study had dose levels below 100 ppm, further analysis was conducted to establish stability in the diet at 1 ppm. Stability at 1 ppm in the test diet was established at room temperature for 8-days and under cold, dark and sealed conditions for 5 weeks (study no. IET 05-5020, 2007).

Chemical analysis for homogeneity and concentration of the test substance in the diet was performed for each dose level on samples (approximately 50 g each) taken from the top, middle, and bottom portions of the mixer at the first preparation and at 3, 6, 9 and 12 months. At each analysis, a sample was also taken from the control diet and analysed to verify that the diet was not contaminated by the test substance.

At chemical analysis, extracts of the test substance from samples were obtained by addition of water and sulfuric acid followed by further addition of acetonitrile, and analysed by high performance liquid chromatography using a LC-2000 Plus (JASCO Corporation, Tokyo, Japan) with L-column ODS (Chemical Evaluation and Research Institute, Tokyo, Japan).

The coefficient of variation (CV) of the test substance concentration in the diet for each dose level was within 12.5% and satisfied the acceptable limit of 15%, confirming homogeneity of the test substance in the diet. Mean concentrations of the test substance in test diets determined on the samples at nominal levels of 1, 4, 20 and 2000 ppm were 1.1 ± 0.06, 3.8 ± 0.15, 20 ± 1.0 and 2040 ± 29.9 ppm (Mean ± S.D., N=3), respectively. The values were 95% - 110% of the target concentrations and within the acceptable limit of 100% ± 10% of the target concentrations). There were no detectable levels of the test substance in the control diet samples, indicating that the diet was not contaminated with the test substance. The residual concentrations of the test substance in test diets after use were determined twice during the treatment period for all dose levels. The concentrations found in the diets after use were 95% - 110% of the target values. Therefore, it was indicated that there were no issues in the preparation method and storage condition of diets in the study.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

Continuously (via diet)

Dose / conc.:

1 ppm

Remarks:

equivalent to 0.0247 mg/kg bw/day (males) and 0.0255 mg/kg bw/day (females)

Dose / conc.:

4 ppm

Remarks:

equivalent to 0.102 mg/kg bw/day (males) and 0.102 mg/kg bw/day (females)

Dose / conc.:

20 ppm

Remarks:

equivalent to 0.515 mg/kg bw/day (males) and 0.514 mg/kg bw/day (females)

Dose / conc.:

2 000 ppm

Remarks:

equivalent to 53.5 mg/kg bw/day (males) and 53.6 mg/kg bw/day (females)

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
In the preceding 90-day dietary study (M-398722-01-2, 2007), four males and four females were given the test substance by incorporating it into the basal diet at levels of 0, 20, 200, or 2000 ppm. Ophthalmological effects, characterised by degeneration of corneal epithelial cells was seen at all dose levels tested. Urinalysis also showed effects, characterised by lower pH at each dose level and a positive ketone reaction at 2000 ppm. At 2000 ppm there were also effects on body weight gain, organ weights and haematology parameters. As a NOAEL was not determined in the 90-day study, dose levels of 1 and 4 ppm were selected for the chronic study, in order to include dose levels lower than the low dose in the 90-day study. 20 ppm, which was the lowest dose in the 90-day study was set as the middle-high dose, and 2000 ppm, at which toxicity other than ocular effects or urine alterations was expected, was selected as the high dose level.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on weekdays and once a day on weekends and holidays
- Cage side observations: Mortality/viability and general clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
- Parameters checked:
Home-cage: Spontaneous motor activity, abnormal posture/position, abnormal behaviour / stereotypical behaviour, tremors, convulsions (tonic, clonic)

Social behavior during removal from cage: Sociability (friendly, indifferent, aggressive)

Open field: Spontaneous motor activity (including exploring behaviour), abnormal posture / position, abnormal behaviour/stereotypical behaviour, tremors, convulsions, abnormal gait (including motor coordination), respiration, changes in skin and fur (e.g., piloerection), changes in eye ball, changes in palpebra (existence or nonexistence of closure), changes in pupil size (contracted, dilated), lacrimation, salivation, discharge (e.g., ocular, auricular, nasal, and vaginal discharges), conjunctival and oral mucosa (anemic, hyperemic), abnormal vocalization, defaecation, urination, response to sharp noise and response to touch stimulation

Palpation: Integument (abnormalities of callosity, claw), muscle (development, tension)

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly from Weeks 1 to 13, and every 4 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation and at Weeks 13, 26 and 52
- Dose groups that were examined: All dose groups
- Parameters assessed: Eyeball, eyelid, conjunctiva, cornea, anterior chamber, pupil, iris, lens, vitreous body and fundus

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to study initiation and at Weeks 13, 26 and 52
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Haematocrit (Ht), haemoglobin concentration (Hb), erythrocyte count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), reticulocyte count (Retics), prothrombin time (PT), activated partial thromboplastin time (APTT), total leucocyte count (WBC), differential leucocyte count [lymphocyte, neutrophil, monocyte, eosinophil, basophil and Large unstained cell]

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to study initiation and at Weeks 13, 26 and 52
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGTP), creatinine (Creat), blood urea nitrogen (BUN), total protein (TP), albumin (ALB), globulin (Glob), albumin/globulin ratio (a/g ratio), glucose (Gluc), total cholesterol (T.chol), triglyceride (TG), total bilirubin (T.bil), calcium (Ca), inorganic phosphorus (P), sodium (Na), potassium (K) and chloride (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to study initiation and at Weeks 13, 26 and 52
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Fresh urine collected from trays, specific gravity, urobilinogen, protein, pH, occult blood, ketones, bilirubin, glucose and appearance. 24-hour urine samples collected using urine receptacles - urine volume and urinary sediments

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, the whole body of each animal was examined carefully, and all gross findings were recorded

Bone marrow samples from sternum were obtained from all animals at terminal necropsy after 52 weeks of treatment and nucleated cell counts were determined using Advia 120. In addition, smears of the bone marrow were prepared and stained with May-Grünwald and Giemsa.

Organ weight measurement was performed on all animals at the scheduled kill after 52 weeks of treatment. The following organs were removed at necropsy and their weights (absolute organ weights) were recorded before fixation. Bilateral organs were measured at each side, and then calculated total weight. Relative organ weights were calculated as ratios of organ weights to final body weight. Brain, pituitary, thyroids with parathyroids, heart, thymus, liver with gallbladder, kidneys (bilateral), spleen, adrenals (bilateral), testes (bilateral), epididymides (bilateral), prostate, ovaries (bilateral), uterus

The following organs and tissues were removed from all animals at necropsy, and fixed in 10% neutral-buffered formalin. The eyes were fixed in a phosphate-buffered mixed solution of formalin and glutaraldehyde (pH 7.2) for about 3 days and thereafter transferred into neutral-buffered 10% formalin. The testes were fixed in a mixed solution of formalin, sucrose, and acetic acid (FSA solution) for about 5 or 6 days and then transferred into neutral-buffered 10% formalin. Brain (cerebrum, cerebellum, pons, and medulla oblongata), spinal cord (cervical, thoracic, and lumbar regions), sciatic nerve (unilateral, close to muscle), pituitary, thymus, thyroids with parathyroids (bilateral), adrenals (bilateral), tonsils, spleen (middle and caudal regions), bone with bone marrow (sternum; femur, unilateral; and rib), lymph nodes (cervical and mesenteric), heart (left and right ventricular walls and interventricular septum including valve), aorta, tongue, buccal mucosa of oral cavity, pharynx, salivary glands (submaxillary and parotid), oesophagus, stomach (cardiac, fundic, and pyloric regions), liver (left and right lateral lobes and hilus), gallbladder, pancreas, duodenum, jejunum, ileum (including peyer’s patches), caecum, colon, rectum, nasal cavity, larynx, trachea, lung (hilus of right lung including mainstem bronchi and left and right caudal lobes), kidneys (bilateral), urinary bladder, testes (bilateral), epididymides (bilateral), prostate, penis, ovaries (bilateral), oviducts (bilateral), uterus (horns, corpus, and cervix), vagina, diaphragm, eyes (with retina and optic nerve, bilateral), lacrimal glands (bilateral), femoral muscle (unilateral), skin (lumbodorsal region), mammary gland (abdominal region) and all gross lesions

HISTOPATHOLOGY: Yes, histopathological examination was performed on the following organs from all animals. Microscopic examination was performed on preparations stained with haematoxylin and eosin. Brain (cerebrum, cerebellum, pons, and medulla oblongata), spinal cord (cervical, thoracic, and lumbar regions), sciatic nerve (unilateral ,close to muscle), pituitary, thymus, thyroids (bilateral), parathyroids (bilateral), adrenals (bilateral), spleen (middle and caudal regions), bone with bone marrow (sternum; femur, unilateral; and rib), lymph nodes (cervical and mesenteric), heart (left and right ventricular walls and interventricular septum including valve), aorta, pharynx, salivary glands (submaxillary and parotid), oesophagus, stomach (cardiac, fundic, and pyloric regions), liver (left and right lateral lobes and hilus), gallbladder, pancreas, duodenum, jejunum, ileum (including peyer’s patches), caecum, colon, rectum, nasal cavity, larynx, trachea, lung (hilus of right lung including mainstem bronchi and left and right caudal lobes), kidneys (bilateral), urinary bladder, testes (bilateral), epididymides (bilateral), prostate, ovaries (bilateral), uterus (horns, corpus, and cervix), vagina, eyes (with retina and optic nerve, bilateral), lacrimal glands (bilateral), femoral muscle (unilateral), skin (lumbodorsal region), mammary gland and all gross lesions

Statistics:

Statistical significance was estimated at 5 and 1% levels of probability.

The data of body weight, urine specific gravity, urine volume, haematological parameters, blood biochemical parameters, and organ weights were evaluated by Bartlett’s test for equality of variance. When group variances were homogeneous, a parametric analysis of variance of a one way layout type was conducted to determine if any statistical differences existed among groups. When the analysis of variance was significant, Dunnett’s multiple comparison test was applied to determine significant differences between treated groups and control group. When the group variances were heterogeneous, the data were evaluated by Kruskal-Wallis non-parametric analysis of variance. When significant, Dunnett type mean rank sum test was applied to determine significant differences between treated groups and control group.

The data on scored clinical signs, food consumption, and urinalysis parameters (except urine specific gravity and volume) were evaluated by Kruskal-Wallis analysis. When significant difference among group means was indicated by this non-parametric procedure, Dunnett type mean rank test was applied to determine significant differences between treated groups and control group.

Fisher’s exact probability test (one-tail analysis) was used to analyze mortality, and the data of general clinical signs, ophthalmology, and gross pathology, and histopathology.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2000 ppm:
Three females showed soft faeces. Mucosal and mucosal bloody faeces were also observed in two of these three females. For one of these animals, mucosal bloody faeces and mucosal faeces were repeatedly observed, the former was recorded 12 times and the latter three times during the treatment period. Animals at the other dose levels did not show faecal abnormalities. The findings were considered to be treatment related because they were not observed in these dogs in the acclimatization period, nor were they seen in control dogs during treatment. However, the cause of these finding was unclear as there were no corresponding gross or histopathological abnormalities detected in the large intestine after 52 weeks of treatment.

Opacity of the eye was observed in two males (findings were bilateral in one animal), starting at Weeks 2 and 9 respectively. This was sustained until the end of treatment. A summary of all ocular lesions is provided in Table 5, in the field "Any other information on results incl. tables".

All males and one female showed interdigital swelling in fore and/or hind limbs, this was statistically significant in males as no control animals showed the same lesions. The cause of the lesion was compression by the wire mesh of the cages.

Other findings at 2000 ppm included wounds, hair loss, vomiting feed and bloody discharge from the vagina, however, there was no correlation to the findings and they were not considered treatment related.

20 ppm:
One male and one female showed opacity of the eye. This was observed from Week 3 in both dogs, but had reversed by Weeks 17 and 19 respectively.

Two females showed interdigital swelling in fore and/or hind limbs. The cause of the lesion was compression by the wire mesh of the cages.

The cause of the interdigital lesions seen at 20 and 2000 ppm was not considered to be treatment related, as there were no corresponding effects such as debility or ataxia which might contribute to compression on the wire mesh floor. Additionally, this is a finding that has been seen in control dogs in other chronic toxicity studies.

4 ppm:
One male showed opacity of the eye, this occurred from Week 22 and persisted until the termination of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Individual body weight gains throughout the treatment period are shown in Table 1, in the field "Any other information on results, incl. tables".

2000 ppm:
Mean body weights at Week 52 were 95% in males and 91% in females of the respective controls. Body weight gains throughout the treatment period were 0.1 kg and 0.2 kg in males and females, respectively, and tended to be low (but not statistically significant) when compared to the controls (males, 0.6 kg; females, 0.9 kg).

Body weights in males and females treated at 20 ppm or less were comparable to the respective controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Average daily test substance intake (mg/kg bw/day) were as follows:

1 ppm: 0.0247 mg/kg bw/day (males) and 0.0255 mg/kg bw/day (females)
4 ppm: 0.102 mg/kg bw/day (males) and 0.102 mg/kg bw/day (females)
20 ppm: 0.515 mg/kg bw/day (males) and 0.514 mg/kg bw/day (females)
2000 ppm: 53.5 mg/kg bw/day (males) and 53.6 mg/kg bw/day (females)

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

2000 ppm:
Two males showed corneal opacity at Weeks 13, 26 and 52. The corneal lesion in one male was observed in left side only at Week 13, but it became bilateral at Weeks 26 and 52. For the other male, corneal opacity was only present in the left eye at each examination point. One female showed corneal opacity in the right eye at Weeks 13 and 26, but the lesion was not present at Week 52.

20 ppm:
One male showed corneal opacity in the right eye at Weeks 13 and 52. Two females showed corneal opacity, the first at Week 13 and 26 in the left eye, but not at Week 52, the second in the right eye at Week 52.

4 ppm:
One male showed corneal opacity in the right eye at Week 26 and 52. There were no abnormalities in the females.

1 ppm:
No abnormalities were observed in males or females.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant changes observed in treated groups compared to the control group are shown in Table 2, in the field "Any other information on results, incl. tables".

2000 ppm:
In the haematological examination, decreases in haematocrit, haemoglobin concentration, mean corpuscular volume and mean corpuscular haemoglobin were noted in males and females at each examination point, and induction of microcytic hypochromatic anaemia by the treatment was indicated. In addition, increases in platelet count in males and females and erythrocyte count in females were considered to indicate increased haematopoiesis as a compensatory reaction to the anaemia.

For blood coagulation related parameters, activated partial thromboplastin time was significantly prolonged in males of the 2000 ppm group at week 52. In the historical background data, activated partial thromboplastin time in males at Week 52 was 10.3 ± 0.3 sec (N=16). The values of the parameter in males of the 2000 ppm and control groups in the present study were 10.7 ± 0.3 sec and 9.6 ± 0.3 sec, respectively. Therefore, it was concluded that the statistical significance noted in the prolongation in activated partial thromboplastin time in the high dose group of the present study was meaningless, because it had occurred due to the short time of this parameter in the control group.

In males of the 2000 ppm group, total leucocyte count was significantly increased at Week 52 and neutrophil count was also significantly increased at the examination point. When individual animal data was assessed, total leucocyte and neutrophil counts in Animal No. 17 were extremely high. Males treated at 2000 ppm other than Animal No.17 showed higher values than those in the control before initiation, but their changes throughout the treatment period were comparable to the control. Therefore, it was considered that the increases in total leucocyte and neutrophil counts had occurred due to the high values in Animal No.17, and there were no abnormalities in the other three males. Histopathologically, increased haematopoiesis mainly attributed by granulocyte was observed in the bone marrow of Animal No. 17, the cause of the increased haematopoiesis was considered to be the interdigit swelling observed clinically. Therefore, the increases in total leucocyte and neutrophil counts were considered not to be treatment-related. For other changes in differential leucocyte count, significant increases in monocyte in males of the 2000 ppm group at week 52 and eosinophil in females of the group at Weeks 13 and 52 were noted. As the numbers of these cells in differential leucocyte count were extremely small, the difference of actual numbers with those in the control group was extremely small. Therefore, the changes were considered to be not toxicologically significant.

No significant effects were observed at the other dose levels.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant changes observed in treated groups compared to the control group are shown in Table 3, in the field "Any other information on results, incl. tables".

2000 ppm:
Males showed a significant increase in alkaline phosphatase at Weeks 26 and 52. Although no abnormal organ weight was detected in the present study, an increase in the liver weight which considered to be treatment-related was noted in males and females of the 2000 ppm group of the previous repeated 90-day oral toxicity study (M-398722-01-2, 2007). Therefore, the increased alkaline phosphatase observed in the present study was considered to be related to treatment.

At Week 52, significant increases in total protein and globulin, and a significant decrease in albumin / globulin ratio were noted in males. As all males in the 2000 ppm group showed interdigit swelling clinically, it was considered to be highly possible that the inflammatory lesion caused the elevation of immunoglobulin. Therefore, it was considered that these changes in proteins were incidental and not treatment-related.

For blood electrolytes, a decrease in chloride in males and an increase in calcium in females were noted in the 2000 ppm at Week 52.

20 ppm:
Significant decreases in creatinine in males and females at Weeks 13 and 26, and blood urea nitrogen in males at Week 52 were noted. Females showed an increase in calcium at Week 52.

The significant decreases in creatinine, blood urea nitrogen and/or total bilirubin in males and females of the 20 and 2000 ppm groups were considered to have no toxicological significance. When compared with the historical controls, chloride in males and calcium in females of the control group in the present study were considerably high and low, respectively. Electrolyte values in the treated groups were close to the mean value of the historical control. Therefore, it was considered that the significant changes in these electrolytes had no toxicological significance (electrolyte historical control data is presented in Table 4 in the field “Any other information on results incl. tables”).

4 ppm:
Males showed a significant increase in albumin at Week 26. There were no significant findings in females when compared with the values in each group before initiation of treatment or in the corresponding controls.

1 ppm:
There were no significant findings in males and females when compared with the values in each group before initiation of treatment or in the corresponding controls.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

2000 ppm:
One male and female showed ketone positive reactions at all examination points. In addition, positive reaction for ketone was observed in two males at Weeks 13 and 26, respectively, and one female at Weeks 26 and 52. There were no dogs showing a ketone positive reaction in the control group at any examination points.

Low urine pH (<7.0) was observed in three females, the first at Weeks 13 and 52, the second at Week 26 and the third at Week 52. These values tended to be lower than the controls and the values noted before study initiation. Additionally, at Week 13 the pH values for the females were statistically significantly lower when compared to controls.

20 ppm:
Two males and two females showed ketone positive reactions at Week 52.

Low urine pH (<7.0) was observed in two females at Week 52. These values tended to be lower than the controls and the values noted before study initiation.

At 1 and 4 ppm, no significant changes were observed.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes observed in the treated groups compared to the control group are shown in Table 6, in the field "Any other information on results, incl. tables".

2000 ppm:
Significant increases in absolute and relative weights of the kidneys were noted in males. Although histopathological examination failed to detect any abnormalities in the kidney, the weight change was considered to be treatment-related, because both absolute and relative weights were increased.

20 ppm:
A significant decrease in relative weight of the epididymides in males and a significant increase in relative weight of the spleen in females were noted. However, these changes did not show a dose response as they were not seen at 2000 ppm.

1 ppm:
A significant increase in absolute and relative weights of the ovaries was noted, however, these changes did not show a dose response as they were not seen at 20 or 2000 ppm.

There were no significant changes in organ weight in males and females at 4 ppm.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

2000 ppm
Two males showed opacity of the eye.

One male showed swelling of the skin, the skin lesion occurred in the paw of fore limb and corresponded to the interdigit swelling recorded in the clinical observations.

Other gross findings included, enlargement of the lymph nodes in two males (cervical and iliac lymph nodes, respectively). Thickening of the small intestine and a spot on the thyroid in one female.

4 ppm:
One male showed opacity of the eye.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

2000 ppm
Two males and one female showed degeneration of corneal epithelial cells of the eye. The lesion was characterized by loss of corneal epithelial cells and appearance of large cells with clear cytoplasm at the basal portion of the cornea. In addition, fine intracytoplasmic vacuole was observed in a few populations of the degenerated corneal epithelial cells. The grade of the lesion was slight in all animals, and the laterality was unilateral in two animals (one male and one females) and bilateral in the other male.

Two males showed an anterior cyst of the pituitary and fatty change in zona reticularis of the adrenal. As these lesions were also observed in one dog of the control group, they were not considered to be treatment related.

Other histopathological findings observed at 2000 ppm were as follows:
- Granuloma of the skin and lymphadenitis in the cervical lymph node in one male.
- Increased haematopoiesis in the sternal and femoral bone marrow in one male.
- Lymphocyte hyperplasia in the iliac lymph node in one male.
- C-cell hyperplasia of the thyroid in one female.

Among these histopathological lesions, granuloma of the skin was correspondent to the inter-digit swelling observed clinically. Increased haematopoiesis in sternal and femoral bone marrow was observed in the previously conducted repeated 90-day oral toxicity study in dogs (M-398593-01-2, 2007), and was attributed to an increase in erythroblasts that was considered to be related to anaemia induced by the treatment. However, in this study granulocytic cells were the main component of the increased haematopoiesis. The increased haematopoiesis in the bone marrow, and lymphadenitis and lymphocyte hyperplasia of the cervical and iliac lymph nodes, respectively, were considered to be secondary changes subsequent to inflammatory lesions. All dogs with these histopathological findings showed inter-digit swelling clinically, and there were no inflammatory changes in other organs examined. Lymph fluid flows into the cervical (superficial cervical) lymph node directly from fore limb and into the iliac lymph node from hind limb via the popliteal and inguinal lymph nodes. Therefore, it was suspected that the lesions were secondary to the inter-digit swelling which was observed frequently in males of the dose group. One female showed C-cell hyperplasia in the thyroid, but it was considered to be incidental and not treatment-related, because no other dogs in the 2000 ppm group showed any histopathological abnormalities in the thyroid.

20 ppm
One male showed degeneration of corneal epithelial cells of the eye. The lesion was characterized by loss of corneal epithelial cells and appearance of large cells with clear cytoplasm at the basal portion of the cornea. In addition, fine intracytoplasmic vacuole was observed in a few populations of the degenerated corneal epithelial cells. The grade of the lesion was slight and occurred unilaterally.

4 ppm:
One male showed corneal opacity at necropsy, but the corresponding histopathological lesion was slight erosion/ulcer in the superficial layer of the cornea, but did not include degeneration of corneal epithelial cells, which was the characteristic morphology observed in dogs treated at 20 ppm or more. Therefore, it was highly possible that the eye lesion observed in the single animal at 4 ppm was incidental and not treatment-related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Effect level:

4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen.

Remarks on result:

other: equivalent to 0.102 mg/kg bw/day (males and females)

Key result

Dose descriptor:

LOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

ophthalmological examination

urinalysis

Remarks on result:

other: equivalent to 0.515 and 0.514 mg/kg bw/day (in males and females, respectively). The ocular findings are concluded not to be relevant to humans.

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Body Weight

Table 1: Group mean body weight at Week 52 and group mean body weight gains throughout the treatment period.

Dose (ppm)	Male		Female
	Mean body weight (kg)	Body weight gain (kg)	Mean body weight (kg)	Body weight gain (kg)
0	10	0.6	10.4	0.9
1	10.3	0.9	10.0	0.7
4	9.9	0.4	10.1	0.8
20	9.9	0.5	10.2	0.9
2000	9.5	0.1	9.5	0.2

 Haematology

 Table 2: Summary of the haematology data relative to the control (%)

Sex and dose level (ppm)		Males				Females
Parameter	Week	1	4	20	2000	1	4	20	2000
Haematocrit	-1	99	98	98	102	102	107	105	98
	13	102	103	97	92^*	100	101	101	89^*
	26	100	107	98	90^*	101	101	104	90
	52	102	102	98	88	103	98	102	90
Haemoglobin concentration	-1	99	100	99	103	101	106	105	96
	13	101	103	97	91^*	99	100	100	87^*
	26	99	104	97	88^*	100	101	103	87^*
	52	102	102	98	88^**	103	98	102	88^*
Erythrocyte count	-1	98	97	99	103	103	106	104	99
	13	100	101	101	110	101	101	105	114
	26	98	104	101	110	102	101	105	118^**
	52	100	101	101	109	105	99	103	116^*
Mean Corpuscular volume	-1	101	101	98	99	99	101	101	98
	13	102	103	96	84^**	99	100	96	78^**
	26	102	103	97	82^**	99	100	99	76^*
	52	102	102	97	81^**	98	100	99	78^**
Mean corpuscular haemoglobin	-1	101	103	100	100	98	100	101	97
	13	101	103	97	83^**	98	99	96	76^**
	26	100	100	96	81^*	98	100	98	74
	52	101	102	96	81	98	99	99	76^*
Platelet count	-1	87	94	87	106	99	88	92	97
	13	94	105	124	162^**	94	93	102	143^**
	26	93	108	121	164^**	97	100	106	142^**
	52	90	106	109	158^**	113	94	106	134
Activated partial thromboplastin time	-1	106	108	105	108	97	96	100	102
	13	103	99	103	104	98	99	100	101
	26	102	100	102	107	97	99	102	100
	52	105	100	104	111^**	94	94	97	97
Total leucocyte count	-1	118	122	115	128	97	95	108	107
	13	105	103	108	120	91	98	110	107
	26	109	115	127	131	90	99	117	128
	52	114	143	145	194^*	95	92	114	113
Differential leucocyte count:
Neutrophil	-1	118	126	131	137	85	83	93	101
	13	102	96	108	119	82	95	97	108
	26	105	111	138	132	79	93	106	130
	52	113	160	165	217^*	93	86	104	109
Monocyte	-1	104	96	96	100	93	105	109	121
	13	108	91	92	132	83	117	117	125
	26	117	110	123	150	85	117	120	146
	52	116	149	140	242^**	114	126	154	163
Eosinophil	-1	130	96	109	91	193	227	147	187
	13	95	90	134	141	135	135	208	285^*
	26	113	100	125	134	179	179	183	304
	52	158	127	154	173	121	143	164	293^**
Basophil	-1	100	100	100	100	100	113	100	63
	13	120	100	80	80	75	75	75	38
	26	100	100	100	100	67	67	67	44^*
	52	120	100	120	100	75	63	75	50

Figures show the ratio (%) to the control (100%) at the examination period.

* p ≤ 0.05; ** p ≤ 0.01 (Dunnett’s multiple comparison test).

Clinical Biochemistry

Table 3: Significant changes observed in the treated groups compared to the control group (%).

Sex and dose level (ppm)		Males				Females
Parameter	Week	1	4	20	2000	1	4	20	2000
Alkaline phosphatase	-1	97	97	102	103	109	106	102	99
	13	82	75	99	123	135	123	120	103
	26	101	89	120	182^**	162	158	154	116
	52	89	88	124	174^*	158	203	193	144
Creatinine	-1	94	96	97	91	97	99	99	96
	13	93	94	78^*	72^**	92	88	78^**	77^**
	26	98	90	76^*	67^**	95	92	80^*	80^*
	52	97	87	74	65^*	100	98	83	79
Blood urea nitrogen	-1	94	92	93	93	88	91	96	95
	13	96	107	95	94	81	87	83	93
	26	97	116	90	76	84	82	78	89
	52	90	94	75^**	68^**	96	113	83	85
Total protein	-1	98	95	100	100	101	102	101	102
	13	101	94	105	108	100	106	103	103
	26	103	99	107	113	100	105	106	101
	52	102	97	111	122^**	104	106	112	109
Albumin	-1	98	98	102	102	102	103	102	102
	13	103	103	104	100	103	104	104	102
	26	103	107^*	106	101	104	106	105	102
	52	102	103	103	96	105	107	107	105
Globulin	-1	98	92	97	97	101	100	100	103
	13	100	87	106	117	97	107	103	105
	26	103	93	108	124	98	103	106	99
	52	102	92	119	144^**	103	104	116	112
Albumin/globulin ratio	-1	99	106	105	104	101	103	103	99
	13	101	119	97	85	106	99	102	96
	26	98	114	98	80	107	103	103	102
	52	98	112	85	66^*	101	102	97	93
Total billirubin	-1	89	100	89	111	69	62	77	62
	13	89	133	89	78	71	64	64	57^*
	26	78	122	89	67	77	77	69	54
	52	82	109	73	55^**	87	80	67	53^**
Calcium	-1	101	99	101	101	102	103	101	103
	13	104	103	104	106	102	103	102	104
	26	103	103	103	103	100	102	101	103
	52	101	101	101	104	101	103	105*	106^**
Chloride	-1	101	100	101	101	101	100	100	101
	13	100	100	100	100	101	100	100	102
	26	100	100	100	99	101	101	100	101
	52	100	101	100	98^*	101	100	100	100

Figures show the ratio (%) to the control value (100%) at the examination period.
* p ≤ 0.05; ** p ≤ 0.01 (Dunnett’s multiple comparison test).

Table 4: Comparison of the mean values in chloride and calcium with the historical background data

	Electrolytes
	Chloride (Males)	Calcium (Females)
Historical background data	115.5 ± 1.7 mg/dL	9.8 ± 0.4 mEq/dL
Current study data
0 ppm	117.0 ± 1.3 mg/dL	9.4 ± 0.2 mEq/dL
20 ppm	-	9.9 ± 0.2 mEq/dL
2000 ppm	114.7 ± 1.9 mg/dL	10.0 ± 0.2 mEq/dL

Examination point was after 52 weeks of treatment.
Number of data in the historical control was 16, numbers in the table represent mean ± SD.
-: No significant changes

Eye Lesions

Table 5: Summary of eye lesions

Sex	Dose(ppm)	Animal No.	Clinical observation	Ophthalmology	Necropsy	Histopathology
M	4	9	22-tk (R)	26, 52 (R)	Opacity, cornea (R)	Erosion/ulcer (U)
	20	13	3-19 (R)	13, 52 (R)	NAD	Degeneration, corneal epithelial cell (U)
	2000	18	9-18 (L) 19-tk (B)	13 (L) 26, 52 (B)	Opacity, cornea (B)	Degeneration, corneal epithelial cell (B)
		20	2-tk (L)	13, 26, 52 (L)	Opacity, cornea (L)	Degeneration, corneal epithelial cell (U)
F	20	33	3-17 (L)	13, 26 (L)	NAD	NAD
		34	NAD	52 (R)	NAD	NAD
	2000	38	NAD	13, 26 (R)	NAD	Degeneration, corneal epithelial cell (U)

M, males; F, females; tk, terminal kill after 52 weeks of treatment; (R), right; (L), left; (U), unilateral; (B), bilateral; NAD, no abnormalities detected.
Findings in general clinical observation and ophthalmology were opacity and corneal opacity, respectively; numbers represent treatment week.

Organ Weights

Table 6: Summary of organ weights

Organs	Sex and dose level (ppm)
	Male				Female
	1	4	20	2000	1	4	20	2000
Kidneys                     Abs. Rel.	103 100	105 107	115 117	128* 136**	116 123	103 108	110 115	113 126
Spleen                       Rel.	114	114	124	124	104	108	142**	117
Epididymides         Rel.	91	96	69**	89	-	-	-	-
Ovaries                      Abs.                                                                                  Rel.	- -	- -	- -	- -	193** 203**	120 123	93 95	97 104

Figures show the ratio (%) to the control value (100%) at the examination period. * p ≤ 0.05; ** p ≤ 0.01 (Dunnett’s multiple comparison test). Abs., absolute weight; Rel., relative weight

Conclusions:

The study was performed under GLP conditions and according to OECD 452. In conclusion, the No Observed Adverse Effect Level (NOAEL) of the test substance in Beagle dogs when administered in the diet over a 52-week period was 4 ppm (equivalent to 0.102 mg/kg bw/day in males and females), as in the 20 and 2000 ppm group adverse effects were noted on the eye and urinary properties, and in the 2000 ppm group depression of body weight gain and adverse effects on blood, liver and kidneys were noted.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Dec 2004 - 02 Dec 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Detailed results tables are missing in the translated version of the study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

adopted 21 Sep 1998

Deviations:

yes

Remarks:

detailed results tables are missing in the translated study report

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KITAYAMA LABES Co., Ltd (Hongo farm, Japan)
- Age at study initiation: 6 months
- Weight at study initiation: 7.5 - 9.5 kg (males), 7.3 - 9.5 kg (females)
- Housing: Animals were housed individually in stainless steal cages (width 835 mm × depth 900 mm × height 800 mm) sustained in racks equipped with automatic sweepers
- Diet: Certified pellet diet DS-A (Oriental Yeast Co., Ltd., Tokyo, Japan), moistened with water
- Water: Well water (ad libitum), pressed through a rapid filtration unit with sand filter and an adsorption unit with charcoal filter, was sterilized with sodium hypochlorite and ultraviolet light
- Acclimation period: 34 days (males), 42 days (females)

DETAILS OF FOOD AND WATER QUALITY:
- Food: The supplier analysed each lot of the basal diet for the following nutrients - moisture, crude protein, crude fat, crude ash, crude fibre and nitrogen-free extract
- Food: The supplier analysed each lot of the basal diet for the following contaminants - Mercury, Cadmium, Lead, Chromium, Arsenic, Selenium, γ-BHC, DDT (and related compounds), Aldrin, Dieldrin, Endrin, Heptachlor, Malathion, Parathion, Aflatoxin (B1, B2, G1 and G2), Polychlorobiphenyl, Estradiol, Dimethylnitrosamine, Diethylnitrosamine
- Water: Analysed for Arsenic, Lead, Total mercury, Cadmium, Selenium, Polychlorinated biphenyl, N-Nitroso compounds, Total trihalomethanes, Simazine, Thiram, Thiobencarb, 1,3-Dichloropropene

Analysis confirmed contaminant level were within the acceptable ranges for food and water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): more than 10 times per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Details on route of administration:

The test substance was incorporated into the basal diet and administered to animals of both sexes continuously for a period of 92 days (13 weeks).

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): The test substance was mixed with part of the basal diet in a pestel and mortar, then this premix was mixed with the rest of the basal diet by means of a blending machine
- Storage temperature of food: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis for homogeneity and concentration of the test substance in diet were performed for each dose level on samples (approximately 50 g each) taken from the top, middle and bottom portions of the mixer at the first (November 30, 2004) and last (March 10, 2005) preparation. At each analysis, a sample was also taken from the control diet and analysed to verify that the diet was not contaminated by the test substance.

At the 8th preparation (January 13, 2005), concentrations of the test substance in diet of each dose level including the control, were also determined on samples (approximately 50 g each) taken after use (January 20, 2005). Analysing concentrations in the samples after use indicated appropriateness of the storage procedures. Since there were no deviations in the results of the above analysis, all these stored samples were discarded after termination of treatment.

Samples of test diet were extracted with a mixture of water/sulfuric acid (99.5:0.5, v/v) and acetonitrile, diluted with a mixture of acetonitrile/sulfuric acid (100:0.1, v/v), and determined by high performance liquid chromatography (HPLC) using a LC-2000 Plus (JASCO Corporation, Tokyo, Japan) with Symmetry C18 column (Waters Co., MA, USA).

Homogeneity of the test substance in test diets was checked for each dose level on samples taken from the top, middle, and bottom portions of the mixer. The coefficient of variation (CV) of the test substance concentration in the diet for each dose level was within 3.2% and satisfied the acceptable limit of 15%.

Mean concentrations of the test substance in test diet determined on the samples at nominal levels of 20, 200 and 2000 ppm were 20, 194 and 1959 ppm (Mean, N=2), respectively. The values were 97 - 100% of the target concentrations and satisfied the acceptable limit of 100 ± 10% of the nominal concentrations.

Concentrations of the test substance in test diets prepared at the 8th preparation after use at nominal levels of 20, 200 and 2000 ppm were within the range of 97 - 100% of the nominal levels. There were no detectable levels of the test substance in the control diet samples, indicating that the diet was not contaminated with the test substance.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Continuously (via diet)

Dose / conc.:

20 ppm

Remarks:

equivalent to 0.564 mg/kg bw/day (males) and 0.591 mg/kg bw/day (females)

Dose / conc.:

200 ppm

Remarks:

equivalent to 5.72 mg/kg bw/day (males) and 5.57 mg/kg bw/day (females)

Dose / conc.:

2 000 ppm

Remarks:

equivalent to 58.6 mg/kg bw/day (males) and 62.1 mg/kg bw/day (females)

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
In a preceding repeated dose 90-day oral toxicity study in dogs (IET 04-0027, 2007), animals were given the test substance by incorporating it into the basal diet at a level of 0, 2000, 5000 or 12000/8000 ppm (12000 ppm until Week 2, followed by 8000 ppm from Week 3). Adverse effects were seen at all doses, including findings in the following parameters:

- Clinical observations: In the 12000 ppm group, males and females showed decreases in spontaneous motor activity, staggering gait and crouching position immediately after the initiation of treatment. Deteriorated general status was also observed two weeks after the treatment. The dosage was changed to 8000 ppm from Week 3, but some signs still continued until 8 weeks of treatment. Even in the animals treated at 5000 ppm, similar signs to animals at 12000 / 8000 ppm were observed, but with lower frequency. In the 2000 ppm group, one male showed a decrease in spontaneous motor activity at Week 8.
- Body weights: Males and females in all treatment groups showed lower body weight gain compared to controls.
- Urinalysis: Some animals of both sexes in all treatment groups showed a ketone-positive reaction. In addition, low urine pH was noted in males at 12000 / 8000 ppm, and males and females at 5000 and 2000 ppm. Bilirubin was detected in the urine of males and females at 12000 / 8000 ppm.
- Ophthalmological findings: Opacity of the cornea was observed in males and females treated at 12000 / 8000 ppm, males at 5000 ppm, and both sexes at 2000 ppm. Degeneration of corneal epithelial cells was observed in some animals of both sexes in all treatment groups. In addition, degeneration of axon in optic nerve was observed in both sexes at dose levels greater than 5000 ppm.
- Haematology: Decreases in haematocrit in both sexes at 12000 / 8000 ppm and females at 5000 ppm, haemoglobin in both sexes at more than 5000 ppm and females at 2000 ppm, erythrocyte indices (mean corpuscular volume, mean corpuscular haemoglobin, or mean corpuscular haemoglobin concentration) in both sexes in all treatment groups. Other changes considered relevant included an increase in proerythroblast count in bone marrow cells, an increase in spleen weight, and darkerned color and enlargement of the spleen in males and females at 12000 / 8000 ppm and females at 5000 ppm. In addition, histopathology revealed deposition of brown pigment in the spleen in both sexes at 12000 / 8000 ppm, congestion in the spleen in both sexes at more than 5000 ppm, increased haematopoiesis in systemic bone marrow in both sexes at more than 5000 ppm and males at 2000 ppm. An increase in platelet count was observed in both sexes at more than 5000 ppm and males at 2000 ppm and an increase in leucocyte count attributed to an increase in neutrophil count was observed in both sexes in all treatment groups.
- Clinical chemistry: Males and females at 12000/8000 ppm showed increase in bilirubin.
- Organ findings: Increases in absolute and relative liver weights were detected in both sexes in every treatment group, and histopathologically, diffuse hepatocellular hypertrophy was observed in females at 12000/8000 ppm. In addition, there were increases in relative kidney weight in males in all treatment groups, and absolute kidney weight in males at 12000/8000 ppm.

Based on the result described above, treatment-related changes were observed in every treatment group; thus, a no-observable-adverse-effect level (NOAEL) was not obtained. Therefore, 2000 ppm was selected as a high dose level and based on this, appropriate mid and low doses selected for the present study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on weekdays and once a day on weekends and holidays
- Cage side observations: Mortality / viability and general clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before initiation of treatment and weekly during the treatment period
- Parameters checked:

Home-cage: Spontaneous motor activity, abnormal posture / position, abnormal behaviour / stereotypical behaviour, tremors and convulsions (tonic, clonic)

Social behaviour during removal from cage: Sociability (friendly, indifferent, aggressive)

Open field: Spontaneous motor activity (including exploring behaviour), abnormal posture / position, abnormal behaviour / stereotypical behaviour, tremors, convulsions, abnormal gait (including motor coordination), respiration, changes in skin and fur (e.g., piloerection), changes in eye ball, changes in palpebra (existence or nonexistence of closure), changes in pupil size (contracted, dilated), lacrimation, salivation, discharge (e.g., ocular, auricular, nasal, and vaginal discharges), conjunctival and oral mucosa (anaemic, hyperemic), abnormal vocalization, defaecation, urination, response to sharp noise and response to touch stimulation

Palpation: Integument (abnormalities of callosity, claw) and muscle (development, tension)

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to initiation of treatment and at Week 13
- Dose groups that were examined: All dose groups
- Parameters assessed: Eyeball, Eyelid, Conjunctiva, Cornea, Anterior chamber, Pupil, Iris, Lens, Vitreous body and Fundus

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to initiation of treatment, Week 7 and 13
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Haematocrit (Ht), haemoglobin concentration (Hb), erythrocyte count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), reticulocyte count (Retics), prothrombin time (PT), activated partial thromboplastin time (APTT), total leucocyte count (WBC), differential leucocyte count (lymphocyte, neutrophil, monocyte, eosinophil, basophil and large unstained cell)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to initiation of treatment, Week 7 and 13
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGTP), creatinine (Creat), blood urea nitrogen (BUN), total protein (TP), albumin (Alb), globulin (Glob), albumin / globulin ratio (A / G ratio), glucose (Gluc), total cholesterol (T.Chol), triglyceride (TG), total bilirubin (T.Bil), calcium (Ca), inorganic phosphorus (P), sodium (Na), potassium (K) and chloride (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to initiation of treatment, Week 7 and 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Fresh urine collected from trays, specific gravity, urobilinogen, protein, pH, occult blood, ketones, bilirubin, glucose and appearance. 24-hour urine samples collected using urine receptacles - urine volume and urinary sediments

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, the whole body of each animal was examined carefully, and all gross findings were recorded

Bone marrow samples from sternum were obtained from all animals sacrificed at termination. Nucleated cell counts in the bone marrow were determined with ADVIA 120.

Organ weight measurement was performed on all animals at the scheduled kill after 13 weeks of treatment. The following organs were removed at necropsy and their weights (absolute organ weights) were recorded before fixation. Relative organ weights were calculated as ratios of organ weights to final body weight.

Brain, pituitary, thyroids with parathyroids, heart, thymus, liver with gallbladder, kidneys (bilateral), spleen, adrenals (bilateral), testes (bilateral), epididymides (bilateral), prostate, ovaries (bilateral) and uterus

The following organs and tissues were removed from all animals at necropsy, and fixed in 10% neutral-buffered formalin. The eyes were fixed in a phosphate-buffered mixed solution of formalin and glutaraldehyde (pH 7.2) for about 3 days and thereafter transferred into neutral-buffered 10% formalin. The testes were fixed in a mixed solution of formalin, sucrose, and acetic acid (FSA solution) for about 5 or 6 days and then transferred into neutral-buffered 10% formalin.

Brain (cerebrum, cerebellum, pons, and medulla oblongata), spinal cord (cervical, thoracic, and lumbar regions), sciatic nerve (unilateral, close to muscle), pituitary, thymus, thyroids with parathyroids (bilateral), adrenals (bilateral), tonsils, spleen (middle and caudal regions), bone with bone marrow (sternum; femur, unilateral; and rib), lymph nodes (cervical and mesenteric), heart (left and right ventricular walls and interventricular septum including valve), aorta, tongue, buccal mucosa of oral cavity, pharynx, salivary glands (submaxillary and parotid), oesophagus, stomach (cardiac, fundic, and pyloric regions), liver (left and right lateral lobes and hilus), gallbladder, pancreas, duodenum, jejunum, ileum (including peyer’s patches), caecum, colon, rectum, nasal cavity, larynx, trachea, lung (hilus of right lung including mainstem bronchi and left and right caudal lobes), kidneys (bilateral), urinary bladder, testes (bilateral), epididymides (bilateral), prostate, penis, ovaries (bilateral), oviducts (bilateral), uterus (horns, corpus, and cervix), vagina, diaphragm, eyes (with retina and optic nerve, bilateral), lacrimal glands (bilateral), femoral muscle (unilateral), skin (lumbodorsal region), mammary gland (abdominal region) and all gross lesions

HISTOPATHOLOGY: Yes, histopathological examination was performed on the following organs from all animals. Microscopic examination was performed on preparations stained with haematoxylin and eosin.

Brain (cerebrum, cerebellum, pons, and medulla oblongata), spinal cord (cervical, thoracic, and lumbar regions), sciatic nerve (unilateral ,close to muscle), pituitary, thymus, thyroids (bilateral), parathyroids (bilateral), adrenals (bilateral), spleen (middle and caudal regions), bone with bone marrow (sternum; femur, unilateral; and rib), lymph nodes (cervical and mesenteric), heart (left and right ventricular walls and interventricular septum including valve), aorta, pharynx, salivary glands (submaxillary and parotid), oesophagus, stomach (cardiac, fundic, and pyloric regions), liver (left and right lateral lobes and hilus), gallbladder, pancreas, duodenum, jejunum, ileum (including peyer’s patches), caecum, colon, rectum, nasal cavity, larynx, trachea, lung (hilus of right lung including mainstem bronchi and left and right caudal lobes), kidneys (bilateral), urinary bladder, testes (bilateral), epididymides (bilateral), prostate, ovaries (bilateral), uterus (horns, corpus, and cervix), vagina, eyes (with retina and optic nerve, bilateral), lacrimal glands (bilateral), femoral muscle (unilateral), skin (lumbodorsal region), mammary gland and all gross lesions

Statistics:

Statistical significance of the difference between the control group and the treated groups was estimated at 5 and 1% levels of probability.

The data of body weight, urine specific gravity, urine volume, haematological parameters, blood biochemical parameters, and organ weights (absolute and relative weights) were evaluated by Bartlett’s test for equality of variance. When group variances were homogeneous, a parametric analysis of variance of a one way layout type was conducted to determine if any statistical differences existed among groups. When the analysis of variance was significant, Dunnett’s multiple comparison test was applied to determine significant differences between treated groups and control group. When the group variances were heterogeneous, the data were evaluated by Kruskal-Wallis non-parametric analysis of variance. When significant, Dunnett type mean rank sum test was applied to determine significant differences between treated groups and control group.

The data on scored clinical signs, food consumption, and urinalysis parameters (except urine specific gravity and volume) were evaluated by Kruskal-Wallis analysis. When significant difference among group means was indicated by this non-parametric procedure, Dunnett type mean rank test was applied to determine significant differences between treated groups and control group.

Fisher’s exact probability test (one-tail analysis) was used to analyze mortality, and the data of clinical sign, ophthalmology, and gross pathology, and histopathology.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 20 ppm, one female showed opacity of the eye from Week 12 to the end of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Individual body weight gains throughout the treatment period are shown in Table 1, in the field "Any other information on results, incl. tables"

At 20 ppm, males showed no noticeable body weight loss during the treatment period. One female showed 0.7 kg loss of body weight. However, this weight loss in the female at 20 ppm was considered incidental, since there were no abnormalities detected in body weight in females at 200 ppm.

At 200 ppm, neither males nor females showed noticeable body weight loss during the treatment period.

At 2000 ppm, three males and three females showed body weight loss during the treatment period, with a range of 0.3 to 1.6 kg.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Average daily test substance intake (mg/kg bw/day) were as follows:

20 ppm: 0.564 mg/kg bw/day (males) and 0.591 mg/kg bw/day (females)

200 ppm: 5.72 mg/kg bw/day (males) and 5.57 mg/kg bw/day (females)

2000 ppm: 58.6 mg/kg bw/day (males) and 62.1 mg/kg bw/day (females)

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

At 20 ppm, opacity of the cornea was noted in two females, one of which also showed vascularization.

At 200 ppm, opacity of the cornea was noted in one female.

At 2000 ppm, ophthalmology revealed opacity of the cornea in two males and one female at Week 13.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A summary of the haemotological data is shown in Table 3, in the field "Any other information on results, incl. tables"

At 20 ppm, males showed significant increases in eosinophil count at Week 7 and 13. This change was, however, considered incidental since it showed no dose response.

At 200 ppm, no significant alterations in haematological parameters were observed in either sex. Although females showed a significant increase in mean corpuscular volume (MCV) (the ratio to the control group was 106%) compared to the haematological examination prior to the start of treatment, it was not considered to be test substance related, since MCV during the treatment period was nearly equivalent to that of the control group and no abnormality was detected.

At 2000 ppm, males and females showed significant decreases in MCV and mean corpuscular haemoglobin (MCH) at Week 7 and 13. Males showed a significant increase in erythrocyte count (RBC) and significant decreases in mean corpuscular haemoglobin concentration (MCHC) and activated partial thromboplastin time (APTT) at Week 13. Females showed significant increases in platelet count (PLT) at Week 7 and 13 and a significant increase in neutrophil at Week 7.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A summary of the clinical biochemistry data is shown in Table 4, in the field "Any other information on results, incl. tables".

At 200 ppm, females showed a significant decrease in total bilirubin (T.Bil) at Week 7. This change was considered incidental because there was no dose response.

At 200 and 20 ppm, males showed a significant increase in alkaline phosphatase (ALP) at Week 7. ALP in males at 2000 ppm was not significantly different when compared with the control, and was equivalent to that of males at 20 ppm. These changes were considered incidental, and not relevant to treatment of the test substance, because they did not show a dose response, and there was no increase in ALP in the animals fed diets at higher dose levels in a preceding repeated dose 90-day oral toxicity study.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At 20 ppm, low urine pH was noted in two females at Week 7 and one male and two females at Week 13, although the changes weren't significant. However, one female showed a urine pH of 7.5 at urinalysis prior to initiation of treatment and no apparent time-dependent decrease afterwards.

At 200 ppm, a significant decrease in urine pH was observed in males at Week 7, low urine pH was observed in one male and two females at Week 7 and 13.

At 2000 ppm, male dogs showed significant decreases in urine pH at Week 7. In addition, urine pH of less than 7.5 was noted in two males at Week 7, one male and two females at Week 13, while a pH of more than 8.0 was noted in most of the animals in the analysis before the initiation of treatment (Week -1) and the analysis of control group during the treatment period. Furthermore, a significant increase in ketone was observed in males at Week 7, ketone-positive reactions were observed in all males and two females at Week 7 and two animals in both sexes at Week 13.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A summary of the organ weight data is shown in Table 2, in the field "Any other information on results, incl. tables".

At 200 and 20 ppm, females showed a significant decrease in absolute brain weight. Males showed no statistically significant alterations in any organs.

At 2000 ppm, males showed no significant alterations in any organs. Females showed a significant increase in relative liver weight. Females also showed a significant decrease in absolute brain weight, however, there were neither effects on relative brain weight nor histopathological changes in the brain in this study. In addition, there were no effects on brain weight in the preceding 90-day oral toxicity study. Thus, changes in absolute brain weights in females across the dose levels were considered incidental with no relevance to treatment of the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 20 ppm, a female showed opacity of the eye. There were no findings in males in the same group.

At 2000 and 200 ppm, type and incidence of the lesions observed in males and females were comparable to those in the control groups.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Bone marrow: a mild increase of haematopoiesis was observed in systemic bone marrow (sternum, femur and rib) in three males and two females at 2000 ppm.

Eye: eosinophilic change and necrosis of corneal epithelial cells were observed in three males and two females at 2000 ppm, one male and one female at 200 ppm, and one male and three females at 20 ppm. Desquamated epithelial cells were also observed in some animals. In addition, fine vacuoles were observed in cytoplasm of degenerated epithelial cells. While corneal epithelium was thinned by decrease of cuboidal cells, it was partly replaced by large cells with clear cytoplasm at the basal layer. These observations were collectively called degeneration of corneal epithelial cells. Although the degree of pathological lesion was mild-to-moderate, frequency, severity, and laterality didn’t have relevance to dose levels. Furthermore, vascularization was also observed in addition to degeneration of corneal epithelial cells in one female at 20 ppm. Inflammatory changes such as infiltration of neutrophils were not observed in any eyes with or without vascularization.

Any other pathological findings were only observed in single animals or were incidental changes with no clear relevance to dose levels.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Effect level:

< 20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Adverse effects seen at all dose levels tested.

Remarks on result:

other: equivalent to <0.564 mg/kg and <0.591 mg/kg bw/day in males and females respectively. The ocular findings are concluded not to be relevant to humans.

Key result

Dose descriptor:

LOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

ophthalmological examination

Remarks on result:

other: equivalent to 0.564 mg/kg and 0.591 mg/kg bw/day in males and females, respectively

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm

System:

eye

Organ:

cornea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

haematopoietic

Organ:

blood

bone marrow

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Body Weight Gains

Table 1: Individual and group mean body weight gains throughout the treatment period (kg)

Dose Level	0 ppm		20 ppm		200 ppm		2000 ppm
	Animal No.	Body weight gain	Animal No.	Body weight gain	Animal No.	Body weight gain	Animal No.	Body weight gain
Males	1	0.8	5	0.6	9	0.2	13	-0.9
	2	0.7	6	-0.2	10	-0.1	14	-1.3
	3	1.0	7	0.5	11	0.3	15	0.9
	4	0.5	8	-0.1	12	0.3	16	-0.8
	Mean	0.8	Mean	0.2	Mean	0.2	Mean	-0.5
Females	21	0.6	25	-0.2	29	0.9	33	-1.5
	22	0.4	26	0	30	1	34	-0.3
	23	0	27	-0.7	31	0	35	0.3
	24	1.1	28	0.2	32	0.4	36	-1.6
	Mean	0.5	Mean	-0.2	Mean	0.6	Mean	-0.8

Organ Weights

Table 2: Summary of organ weight data (relative to controls)

Sex and dose level (ppm)	Males			Females
Parameter	20	200	2000	20	200	2000
Final body weight	94	94	88	92	101	85
Brain (absolute)	93	92	95	92^*	90^*	90^*
Liver (relative)	104	110	109	121	107	123^*

Figures show the ratio (%) to the control value (100%) at the examination period.
* p < 0.05 (Dunnett’s multiple comparison test).

Haematology

Table 3: Summary of the haematology data (relative to controls)

Sex and dose level (ppm)		Males			Females
Parameter	Week	20	200	2000	20	200	2000
Erythrocyte count	13^a	97	105	119^*	97	97	109
Mean Corpuscular volume	7	99	97	86^**	99	101	89^**
	13	98	94	77^**	98	97	75^**
Mean corpuscular haemoglobin	7	99	96	86^**	100	100	87^**
	13	97	92	74^**	98	96	72^**
Mean corpuscular haemoglobin concentration	13	99	99	96^**	101	99	97
Platelet count	7	106	133	144	129	114	229^**
	13	116	154	151	133	122	202^**
Activated partial thromboplastin time	13	97	93	92*	99	101	99
Differential leukocyte count: Neutrophil	7	102	123	163	113	134	186^*
Differential leukocyte count: Eosinophil	7	239^*	113	174	131	161	78
	13	300^*	119	162	105	192	46

Figures show the ratio (%) to the control value (100%) at the examination period.
* p < 0.05, **p < 0.01 (Dunnett’s multiple comparison test). a: week of examination.

Clinical Chemistry

Table 4: Summary of the clinical chemistry data (relative to controls)

Sex and dose level (ppm)		Males			Females
Parameter	Week	20	200	2000	20	200	2000
Alkaline phosphatase	7	140^*	144^*	140	125	112	128
Total bilirubin	7	110	80	110	100	70^*	110

Figures show the ratio (%) to the control value (100%) at the examination period.
* p < 0.05 (Dunnett’s multiple comparison test).

Conclusions:

The study was performed under GLP conditions and according to OECD 409. In conclusion, the No Observed Adverse Effect Level (NOAEL) of the test substance in Beagle dogs when administered in the diet over a 90-day period was less than 20 ppm (equivalent to 0.564 and 0.591 mg/kg bw/day in males and females, respectively), as ophthalmological findings, characterized by degeneration of corneal epithelial cells were seen in all dose levels.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.08 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.6, of Regulation (EC) No 1907/2006.

System:

other: Gastrointestinal tract and endocrine system

Organ:

pancreas

thyroid gland

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

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

May 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

The Wistar rats are one of the standard species and strain used on dermal toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: 340 - 377 g; females: 204 - 255 g
- Fasting period before study: no
- Housing: 5 animals of the same group per cage
- Diet: ad libitum (ssniff® SM R/M “Autoclavable Complete diet for Rats and Mice – Breeding and Maintenance”)
- Water: ad libitum (tap water)
- Acclimation period: 19 - 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.1
- Humidity (%): 31 - 68
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

moistened with sterile water

Details on exposure:

TEST SITE
- Area of exposure: area starting approximately at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank
- % coverage: not less than 10%
- Type of wrap if used: During the exposure period, the test substance was held in contact with the skin with a porous gauze dressing (less than or equal to 8 ply). The test site was further covered with non-irritating tape to retain the gauze dressing and the test substance and to ensure that the animals cannot ingest the test substance.
- Time intervals for shavings or clipplings: approximately 24 hours before the test and repeated as required and as practical depending upon hair growth of indiviual animals and/or effects observed.

REMOVAL OF TEST SUBSTANCE
- Washing: The test substance was gently wiped from the skin with lukewarm water after the 6-hour exposure period, to prevent ingestion.
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10, 100, 1000 mg/kg bw/day
- For solids, paste formed: yes (the test substance was moistened sufficiently with water (no more than 300 μL))


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Remarks:

No dose formulation preparation or analysis was performed during the study, as the test item powder was applied to the rat skin as provided by the Sponsor, without using any vehicle (only moistened with water).

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily for 6 hours, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director at 10, 100 and 1000 mg/kg bw/day, based on the absence of toxicity signs following the acute rat dermal study with tefuryltrione (no deaths and no findings up to a limit dose of 2000 mg/kg/day).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (at the beginning and end of each day)
- Cage side observations: morbidity, mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) on Day 0 and once weekly thereafter

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily after patch removal

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, and at least weekly during treatment period, including before necropsy on day 28 (fasted)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Dose groups that were examined: all (pre-treatment), groups 1 (control) and 4 (high dose) on Day 27 - as no item related findings were noted, no examination was performed for groups 2 and 3.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: RBC (Red Blood Cell (erythrocyte)), Hgb (Haemoglobin concentration), Hct (Haematocrit), MCV (Mean Corpuscular (erythrocyte) Volume), MCH (Mean Corpuscular (erythrocyte) Haemoglobin), MCHC (Mean Corpuscular (erythrocyte) Haemoglobin Concentration), RDW (Red Cell (erythrocyte) volume), Plt (Platelet (thrombocyte) count), MPV (Mean Platelet (thrombocyte) volume), RETIC (Reticulocyte count), WBC (White Blood Cell (leukocyte) count), neutrophils, lymphocytes, monocytes, basophil, eosinophil, large unstained cells, APTT (Activated Partial Thromboplastin Time), PT (Prothrombin Time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: gucose, total bilirubin concentration, urea concentration, cholesterol concentration, triglycerides, creatinine concentration, phosphorus concentration, sodium concentration, potassium concentration, calcium concentration, chloride concentration, total protein concentration, albumin concentration, albumin/globulin ratio, aspartate aminotransferase activity, alanine aminotransferse activity, alkaline phosphatase activity, gamma glutamyltransferase activity, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: Day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: leukocytes, nitrite, pH, protein, glucose, urobilinogen, bilirubin, ketones, blood/erythrocytes, specific gravitiy, sediment, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Day 27
- Dose groups that were examined: all
- Battery of functions tested: grip strength, landing foot splay and fore/hind grip strength, sensory reactivity (e.g. auditory, visual and proprioceptive), modified Irwin test including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation

OTHER: Prior to necropsy, the oestrous cycle of all females was determined by taking vaginal smears.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically.

HISTOPATHOLOGY: Yes: Gross findings, Larynx, Skeletal muscle (quadriceps), Treated skin area, Liver, Skin and subcutis (inguinal), Adrenals, Lungs with bronchi, Small intestine, lymph nodes, Spinal cord, Aorta, Mammary gland (inguinal), Spleen, Brain, Nose/nasal cavity, Sternum with marrow, Epididymides, Ovaries with oviduct, Stomach, Eyes with the optic nerves, Pharynx, Testes, External lachrymal glands, Pancreas, Thymus, Oesophagus, Pituitary, Thyroid with parathyroids, Femur with marrow, Prostate, Tongue, Harderian glands, Salivary glands, Trachea, Heart, Sciatic nerve, Urinary bladder, Kidneys, Seminal vesicles with coagulating glands, Uterus, Large intestine, Vagina

ORGAN WEIGHTS: Brain, Prostate including seminal vesicles with coagulating glands, Adrenals, Epididymides, Ovaries, Heart, Pituitary, Kidneys, Testes, Thyroids with parathyroids, Liver, Thymus, Spleen, Uterus including cervix

Statistics:

From numeric data, the mean and standard deviations values was calculated and statistical analysis was performed. The frequency of clinical observations, and necropsy findings was calculated as applicable.

The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.

Clinical signs:

no effects observed

Description (incidence and severity):

Occasionally slight skin lesions (scab and scar) were observed with similar low incidence across the groups in males (1/10, 1/10, 2/10 and 1/10) and females (2/10, 2/10, 1/10 and 0/10) in the control, low, mid and high dose groups, respectively. Red discharge from eye was observed across the groups in males (3/10, 2/10, 4/10 and 0/10) in the control, low, mid and high dose groups, respectively. Thin fur was seen in one female in Mid dose group. These observations were ascribed to minor effects of friction with the restraint and not related to the test item.

Dermal irritation:

no effects observed

Description (incidence and severity):

At investigation of local effects on the treatment area, no test item related changes were observed on skin. No signs of irritation were noted.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effects considered toxicologically significant were noted on the mean body weight and body weight gain values following daily administration of the test item at dose levels up to and including 1000 mg/kg bw/day, during the treatment period.
In Mid dose males and Low dose female, slightly lower than control body weight gain were noted, sometimes attaining statistical significance.
Based on the isolated incidence and lack of a consistent dose or gender response, these variations were regarded as incidental and without toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse effects at any dose level, all parameters falling with the normal physiological and historical control ranges for the age, strain and sex of rats on test.
Variations, attaining statistical significance were noted in males in higher Mean Platelet Volume (MPV) in Mid and High dose groups (p<0.01). Significant lower levels (p<0.05 and/or 0.01) were noted for Reticulocytes, relative (Retic%) in Low and Mid dose males and for Red Cell Distribution With (RDW) in Mid dose males.
In Low and Mid dose females significantly higher (p<0.05) Red Blood Count (RBC), Haemoglobin concentration (HGB) and Heamatocrit (HCT) level were noted. Significantly higher (p<0.01) Prothrombin time (PTT) and Activated Partial Thromboplastin time (APTT) values were noted in high dose females and in Low dose females, respectively.
Based on the isolated incidence, lack of a consistent dose or gender response and that all values were in the normal historic control range, these variations were regarded as incidental and without toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Albumin (Alb.) concentration was slightly higher than Control in all male dose groups and the differences reached statistical significance (p<0.01). Consequently, Total protein (Tot.Prot) and the Albumin to Globulin ratio (A/G) were also higher, reaching statistical significance (p<0.05 or p<0.01). However, there were no histopathological or other changes associated with these differences at any dose level and females do not show any changes these effects are considered to be non-adverse. As all values in the 10 mg/kg/day group were within the normal historical control range, these changes were considered to be non-adverse at this dose level.
A few other clinical chemistry parameters showed on occasion statistically significant variations, however, there was no dose or gender response and the values were within the physiological ranges. For this reason, these variations were considered not to be toxicologically significant or related to treatment.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects were noted, which could be related to the test item.
Slight differences were recorded in urine pH and specific gravity in High dose group of males and females, respectively, reaching statistical significance (p<0.01 and p<0.05, respectively). There were no statistical differences in urine volume from the control.
The degree of change seen was not sufficient to suggest a toxic effect of test item on urine parameters. In males High dose animals there were higher kidney weights and some evidence of mild histopathological changes so the small urine changes in male High dose animals may be treatment related.
Urine sediment analysis showed similar results to control in all test item treated groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed on Day 27.
Increased vocalization was observed on occasion throughout all the dose groups of males and control, low and high dose females at the modified Irwin test (functional observation battery). However, there were no treatment-related differences compared to the Control group, as the incidence was similar in all groups and no dose, or gender related response were noted, and these variations were considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations.
Slightly increased respiration rate, startle and decreased righting reflex were observed occasionally without relationship to treatment and were in the normal range for all groups, these were considered to be without any toxicologically significance.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In High dose males, slightly higher liver weights (~10%) were measured, reaching statistical significance when adjusted for Body weight (p<0.01) and brain weight (p<0.05). There was a slightly higher liver weight in the Mid dose males, but as the absolute weight was only 1.6% above control, the statistical difference is considered not to reflect a test item-related effect.
In High dose males, slightly higher kidney weights (~10%) were measured, reaching statistical significance when adjusted for Body weight (p<0.01) and brain weight (p<0.05). There was a slightly higher kidney weight in the Low and Mid dose males, but as the values were all within the laboratories historical control range and there was no indication from histopathology of any changes, the statistical difference is considered not to reflect a test item-related effect.
There were no biologically significant differences among groups in the weights of other organs measured, related to body weight or brain (compared to control), although statistically significant variations without trend were noted (i.e brain, prostate, testes, epididymis, thymus, spleen). In the absence of a dose, or gender response, or a consistent correlation with histopathological results, these variations were not considered to reflect an adverse effect.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental gross observations:
Small bilateral adrenal gland was observed in 1/10 Low dose males. In 1/10 High dose male animals, a single firm nodule was seen in right medial lobe in the liver. Small left seminal vesicle with coagulating gland was seen in 1/10 High dose males.
Single clear cyst was observed in 1/10 Control females in the ovary and oviduct and in 1/10 High dose females in the ovary bilateral single dark red focus was observed. Dilatation was observed in the uterine body and horn in 1/10 Control and 1/10 High dose females.
These findings were regarded as incidental, terminal procedure-related or reflected the physiological changes observed during the oestrous cycle in the females.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver, kidneys and exocrine tissue of the pancreas at a dose level of 1000 mg/kg bw/day. Following additional examinations, it was found that the exocrine tissue of the pancreas was also altered at a dose level of 100 mg/kg bw/day.
The lesions in the liver were observed only in males. The renal changes were mostly present in the male gender with the exception of one female discussed below. The microscopic findings in the liver and kidneys were in correlation with organ weight changes in the males. While the pancreas, was proportionally altered in both sexes.
Liver (considered as adaptive response)
Minimal hepatocellular centrilobular hypertrophy was observed in 3/10 High dose males. This change was characterized by enlargement of hepatocytes with granular eosinophilic cytoplasm. No test item-related changes were noted during additional histopathological examination of males from the Mid dose group.
Kidneys
Various test item-related changes were noted in the kidneys including increased eosinophilic droplets, proteinaceous casts, tubular degeneration and pelvic dilatation.
Minimal or mild bilateral increased presence of eosinophilic droplets in the cortical tubules when compared to the Control males, was recorded in 2/10 High dose males. Although eosinophilic droplets are frequently observed in renal tubular epithelium as a common background observation in male rats, there was clear evidence of increase in both size and number of droplets in treated males. Minimal proteinaceous casts, homogeneous, eosinophilic in colour, present in the expanded lumen of tubules of the cortex/corticomedullary regions were seen in 5/10 males and 1/10 females from the High dose groups. Casts were accompanied with minimal tubular degeneration in one of these males. Also, there was increased incidence of minimal unilateral tubular basophilia (3/10) and mild unilateral/bilateral dilatation of renal pelvis (3/10) in High dose males. In Mid dose animals, there was no evidence of lesions associated with administration of the test item in the females. Minimal focal tubular proteinaceous cast at the corticomedullary area of the right kidney was only observed in 1/10 male but this observation was not accompanied with any degenerative change, therefore was considered to be within the normal range, representing a background feature rather than test item effect.
Pancreas
Minimal to moderate increase of zymogen granules of the exocrine acini when compared to untreated controls was microscopically manifested. Affected animals included 2/10 males and 2/10 females from the High dose groups. The greater severity (moderate) was seen in one female. Minimal to moderate acinar necrosis accompanied with mixed cellular inflammatory response in 1/10 High dose males and 2/10 High dose females were also recorded. Moderate degree of necrosis was only seen in one female.
There was a decrease of severity in acinar necrosis or increased zymogen granules from moderate to minimal/mild in the Mid dose groups, moderate degree was not observed. Minimal to mild necrosis was seen in 2/10 males and 2/10 females. Minimal to mild increase of zymogen granules was present in 2/10 males and 3/10 females. Minimal to mild mixed cellular inflammation was noted in 2/10 Mid dose females.
No test item-related findings were microscopically observed at a dose level of 10 mg/kg bw/day.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no test item related changes in the animal oestrous cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrous phases.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

pancreas

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 1: Mean Total Proten, Albumin and Albumin/Globulin ratio on Day 28 with Historical control data Males Dose group (mg/kg bw/day)   control 10 100 1000 Total protein (g/L) Mean 56.66 58.37 61.55** 62.46** Historical control range Min-Max. (mean) 49.60 – 63.90 (56.02) Albumin (g/L) Mean 29.99 31.91** 33.72** 34.45** Historical control range Min-Max. (mean) 26.50 – 35.40 (29.96) Albumin/Globulin ration Mean 1.12 1.21* 1.23** 1.24** Historical control range Min-Max. (mean) 0.90 – 1.40 (1.16) * = p<0.05, ** = p< 0.01 (Duncan’s Multiple Range test)

Table 2: Mean liver weights on Day 28
	Dose groups (mg/kg bw/day)
	control	10	100	1000
Males
Absolute (g)	13.75	13.68	13.97	14.88
	differences in %	-0.5	1.6	8.2
Body weight relative (%)	3.10	3.20	3.31*	3.39**
	differences in %	3.2	6.8	9.4
Brain relative(%)	642	622	659	710*
	differences in %	-3.0	2.6	10.6
Females
Absolute (g)	7.70	7.59	7.32	7.48
	differences in %	-1.4	-5.0	-2.9
Body weight relative (%)	3.10	3.23	3.08	3.15
	differences in %	4.2	-0.6	1.6
Brain relative(%)	384	389	370	382
	differences in %	-1.3	-3.6	-0.5
* = p<0.05, ** = p< 0.01 (Duncan’s Multiple Range test)

Table 3: Mean kidney weights on Day 28
	Dose groups (mg/kg bw/day)
	control	10	100	1000
Males
Absolute (g)	3.07	3.16	3.26	3.34
	differences in %	2.9	6.2	8.8
Body weight relative (%)	0.69	0.74*	0.77**	0.76**
	differences in %	7.2	11.6	10.1
Brain relative(%)	143	144	154	159*
	differences in %	0.7	7.7	11.2
Females
Absolute (g)	1.80	1.74	1.75	1.78
	differences in %	-3.3	-2.8	-1.1
Body weight relative (%)	0.73	0.74	0.74	0.75
	differences in %	1.4	1.4	2.7
Brain relative(%)	90	89	88	91
	differences in %	-1.1	-2.2	1.1
* = p<0.05, ** = p< 0.01 (Duncan’s Multiple Range test)

Conclusions:

The study was performed under GLP conditions and according to OECD 410. Due to the dose-dependent effects found in the exocrine tissue of the pancreas at 100 and 1000 mg/kg bw/day in males and females, a dose level of 10 mg/kg bw/day was considered to be the overall NOAEL for both males and females. No skin irritation was observed up to the highest dose tested, therefore a NOAEL local of 1000 mg/kg bw/day (equivalent to 8 and 5.16 mg/cm²/day for males and females, respectively).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

Species:

rat

Quality of whole database:

The available information comprises one adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

System:

gastrointestinal tract

Organ:

pancreas

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

May 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

The Wistar rats are one of the standard species and strain used on dermal toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: 340 - 377 g; females: 204 - 255 g
- Fasting period before study: no
- Housing: 5 animals of the same group per cage
- Diet: ad libitum (ssniff® SM R/M “Autoclavable Complete diet for Rats and Mice – Breeding and Maintenance”)
- Water: ad libitum (tap water)
- Acclimation period: 19 - 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.1
- Humidity (%): 31 - 68
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

moistened with sterile water

Details on exposure:

TEST SITE
- Area of exposure: area starting approximately at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank
- % coverage: not less than 10%
- Type of wrap if used: During the exposure period, the test substance was held in contact with the skin with a porous gauze dressing (less than or equal to 8 ply). The test site was further covered with non-irritating tape to retain the gauze dressing and the test substance and to ensure that the animals cannot ingest the test substance.
- Time intervals for shavings or clipplings: approximately 24 hours before the test and repeated as required and as practical depending upon hair growth of indiviual animals and/or effects observed.

REMOVAL OF TEST SUBSTANCE
- Washing: The test substance was gently wiped from the skin with lukewarm water after the 6-hour exposure period, to prevent ingestion.
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10, 100, 1000 mg/kg bw/day
- For solids, paste formed: yes (the test substance was moistened sufficiently with water (no more than 300 μL))


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Remarks:

No dose formulation preparation or analysis was performed during the study, as the test item powder was applied to the rat skin as provided by the Sponsor, without using any vehicle (only moistened with water).

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily for 6 hours, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director at 10, 100 and 1000 mg/kg bw/day, based on the absence of toxicity signs following the acute rat dermal study with tefuryltrione (no deaths and no findings up to a limit dose of 2000 mg/kg/day).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (at the beginning and end of each day)
- Cage side observations: morbidity, mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) on Day 0 and once weekly thereafter

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily after patch removal

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, and at least weekly during treatment period, including before necropsy on day 28 (fasted)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Dose groups that were examined: all (pre-treatment), groups 1 (control) and 4 (high dose) on Day 27 - as no item related findings were noted, no examination was performed for groups 2 and 3.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: RBC (Red Blood Cell (erythrocyte)), Hgb (Haemoglobin concentration), Hct (Haematocrit), MCV (Mean Corpuscular (erythrocyte) Volume), MCH (Mean Corpuscular (erythrocyte) Haemoglobin), MCHC (Mean Corpuscular (erythrocyte) Haemoglobin Concentration), RDW (Red Cell (erythrocyte) volume), Plt (Platelet (thrombocyte) count), MPV (Mean Platelet (thrombocyte) volume), RETIC (Reticulocyte count), WBC (White Blood Cell (leukocyte) count), neutrophils, lymphocytes, monocytes, basophil, eosinophil, large unstained cells, APTT (Activated Partial Thromboplastin Time), PT (Prothrombin Time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: gucose, total bilirubin concentration, urea concentration, cholesterol concentration, triglycerides, creatinine concentration, phosphorus concentration, sodium concentration, potassium concentration, calcium concentration, chloride concentration, total protein concentration, albumin concentration, albumin/globulin ratio, aspartate aminotransferase activity, alanine aminotransferse activity, alkaline phosphatase activity, gamma glutamyltransferase activity, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: Day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: leukocytes, nitrite, pH, protein, glucose, urobilinogen, bilirubin, ketones, blood/erythrocytes, specific gravitiy, sediment, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Day 27
- Dose groups that were examined: all
- Battery of functions tested: grip strength, landing foot splay and fore/hind grip strength, sensory reactivity (e.g. auditory, visual and proprioceptive), modified Irwin test including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation

OTHER: Prior to necropsy, the oestrous cycle of all females was determined by taking vaginal smears.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically.

HISTOPATHOLOGY: Yes: Gross findings, Larynx, Skeletal muscle (quadriceps), Treated skin area, Liver, Skin and subcutis (inguinal), Adrenals, Lungs with bronchi, Small intestine, lymph nodes, Spinal cord, Aorta, Mammary gland (inguinal), Spleen, Brain, Nose/nasal cavity, Sternum with marrow, Epididymides, Ovaries with oviduct, Stomach, Eyes with the optic nerves, Pharynx, Testes, External lachrymal glands, Pancreas, Thymus, Oesophagus, Pituitary, Thyroid with parathyroids, Femur with marrow, Prostate, Tongue, Harderian glands, Salivary glands, Trachea, Heart, Sciatic nerve, Urinary bladder, Kidneys, Seminal vesicles with coagulating glands, Uterus, Large intestine, Vagina

ORGAN WEIGHTS: Brain, Prostate including seminal vesicles with coagulating glands, Adrenals, Epididymides, Ovaries, Heart, Pituitary, Kidneys, Testes, Thyroids with parathyroids, Liver, Thymus, Spleen, Uterus including cervix

Statistics:

From numeric data, the mean and standard deviations values was calculated and statistical analysis was performed. The frequency of clinical observations, and necropsy findings was calculated as applicable.

The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.

Clinical signs:

no effects observed

Description (incidence and severity):

Occasionally slight skin lesions (scab and scar) were observed with similar low incidence across the groups in males (1/10, 1/10, 2/10 and 1/10) and females (2/10, 2/10, 1/10 and 0/10) in the control, low, mid and high dose groups, respectively. Red discharge from eye was observed across the groups in males (3/10, 2/10, 4/10 and 0/10) in the control, low, mid and high dose groups, respectively. Thin fur was seen in one female in Mid dose group. These observations were ascribed to minor effects of friction with the restraint and not related to the test item.

Dermal irritation:

no effects observed

Description (incidence and severity):

At investigation of local effects on the treatment area, no test item related changes were observed on skin. No signs of irritation were noted.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effects considered toxicologically significant were noted on the mean body weight and body weight gain values following daily administration of the test item at dose levels up to and including 1000 mg/kg bw/day, during the treatment period.
In Mid dose males and Low dose female, slightly lower than control body weight gain were noted, sometimes attaining statistical significance.
Based on the isolated incidence and lack of a consistent dose or gender response, these variations were regarded as incidental and without toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse effects at any dose level, all parameters falling with the normal physiological and historical control ranges for the age, strain and sex of rats on test.
Variations, attaining statistical significance were noted in males in higher Mean Platelet Volume (MPV) in Mid and High dose groups (p<0.01). Significant lower levels (p<0.05 and/or 0.01) were noted for Reticulocytes, relative (Retic%) in Low and Mid dose males and for Red Cell Distribution With (RDW) in Mid dose males.
In Low and Mid dose females significantly higher (p<0.05) Red Blood Count (RBC), Haemoglobin concentration (HGB) and Heamatocrit (HCT) level were noted. Significantly higher (p<0.01) Prothrombin time (PTT) and Activated Partial Thromboplastin time (APTT) values were noted in high dose females and in Low dose females, respectively.
Based on the isolated incidence, lack of a consistent dose or gender response and that all values were in the normal historic control range, these variations were regarded as incidental and without toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Albumin (Alb.) concentration was slightly higher than Control in all male dose groups and the differences reached statistical significance (p<0.01). Consequently, Total protein (Tot.Prot) and the Albumin to Globulin ratio (A/G) were also higher, reaching statistical significance (p<0.05 or p<0.01). However, there were no histopathological or other changes associated with these differences at any dose level and females do not show any changes these effects are considered to be non-adverse. As all values in the 10 mg/kg/day group were within the normal historical control range, these changes were considered to be non-adverse at this dose level.
A few other clinical chemistry parameters showed on occasion statistically significant variations, however, there was no dose or gender response and the values were within the physiological ranges. For this reason, these variations were considered not to be toxicologically significant or related to treatment.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects were noted, which could be related to the test item.
Slight differences were recorded in urine pH and specific gravity in High dose group of males and females, respectively, reaching statistical significance (p<0.01 and p<0.05, respectively). There were no statistical differences in urine volume from the control.
The degree of change seen was not sufficient to suggest a toxic effect of test item on urine parameters. In males High dose animals there were higher kidney weights and some evidence of mild histopathological changes so the small urine changes in male High dose animals may be treatment related.
Urine sediment analysis showed similar results to control in all test item treated groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed on Day 27.
Increased vocalization was observed on occasion throughout all the dose groups of males and control, low and high dose females at the modified Irwin test (functional observation battery). However, there were no treatment-related differences compared to the Control group, as the incidence was similar in all groups and no dose, or gender related response were noted, and these variations were considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations.
Slightly increased respiration rate, startle and decreased righting reflex were observed occasionally without relationship to treatment and were in the normal range for all groups, these were considered to be without any toxicologically significance.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In High dose males, slightly higher liver weights (~10%) were measured, reaching statistical significance when adjusted for Body weight (p<0.01) and brain weight (p<0.05). There was a slightly higher liver weight in the Mid dose males, but as the absolute weight was only 1.6% above control, the statistical difference is considered not to reflect a test item-related effect.
In High dose males, slightly higher kidney weights (~10%) were measured, reaching statistical significance when adjusted for Body weight (p<0.01) and brain weight (p<0.05). There was a slightly higher kidney weight in the Low and Mid dose males, but as the values were all within the laboratories historical control range and there was no indication from histopathology of any changes, the statistical difference is considered not to reflect a test item-related effect.
There were no biologically significant differences among groups in the weights of other organs measured, related to body weight or brain (compared to control), although statistically significant variations without trend were noted (i.e brain, prostate, testes, epididymis, thymus, spleen). In the absence of a dose, or gender response, or a consistent correlation with histopathological results, these variations were not considered to reflect an adverse effect.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental gross observations:
Small bilateral adrenal gland was observed in 1/10 Low dose males. In 1/10 High dose male animals, a single firm nodule was seen in right medial lobe in the liver. Small left seminal vesicle with coagulating gland was seen in 1/10 High dose males.
Single clear cyst was observed in 1/10 Control females in the ovary and oviduct and in 1/10 High dose females in the ovary bilateral single dark red focus was observed. Dilatation was observed in the uterine body and horn in 1/10 Control and 1/10 High dose females.
These findings were regarded as incidental, terminal procedure-related or reflected the physiological changes observed during the oestrous cycle in the females.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver, kidneys and exocrine tissue of the pancreas at a dose level of 1000 mg/kg bw/day. Following additional examinations, it was found that the exocrine tissue of the pancreas was also altered at a dose level of 100 mg/kg bw/day.
The lesions in the liver were observed only in males. The renal changes were mostly present in the male gender with the exception of one female discussed below. The microscopic findings in the liver and kidneys were in correlation with organ weight changes in the males. While the pancreas, was proportionally altered in both sexes.
Liver (considered as adaptive response)
Minimal hepatocellular centrilobular hypertrophy was observed in 3/10 High dose males. This change was characterized by enlargement of hepatocytes with granular eosinophilic cytoplasm. No test item-related changes were noted during additional histopathological examination of males from the Mid dose group.
Kidneys
Various test item-related changes were noted in the kidneys including increased eosinophilic droplets, proteinaceous casts, tubular degeneration and pelvic dilatation.
Minimal or mild bilateral increased presence of eosinophilic droplets in the cortical tubules when compared to the Control males, was recorded in 2/10 High dose males. Although eosinophilic droplets are frequently observed in renal tubular epithelium as a common background observation in male rats, there was clear evidence of increase in both size and number of droplets in treated males. Minimal proteinaceous casts, homogeneous, eosinophilic in colour, present in the expanded lumen of tubules of the cortex/corticomedullary regions were seen in 5/10 males and 1/10 females from the High dose groups. Casts were accompanied with minimal tubular degeneration in one of these males. Also, there was increased incidence of minimal unilateral tubular basophilia (3/10) and mild unilateral/bilateral dilatation of renal pelvis (3/10) in High dose males. In Mid dose animals, there was no evidence of lesions associated with administration of the test item in the females. Minimal focal tubular proteinaceous cast at the corticomedullary area of the right kidney was only observed in 1/10 male but this observation was not accompanied with any degenerative change, therefore was considered to be within the normal range, representing a background feature rather than test item effect.
Pancreas
Minimal to moderate increase of zymogen granules of the exocrine acini when compared to untreated controls was microscopically manifested. Affected animals included 2/10 males and 2/10 females from the High dose groups. The greater severity (moderate) was seen in one female. Minimal to moderate acinar necrosis accompanied with mixed cellular inflammatory response in 1/10 High dose males and 2/10 High dose females were also recorded. Moderate degree of necrosis was only seen in one female.
There was a decrease of severity in acinar necrosis or increased zymogen granules from moderate to minimal/mild in the Mid dose groups, moderate degree was not observed. Minimal to mild necrosis was seen in 2/10 males and 2/10 females. Minimal to mild increase of zymogen granules was present in 2/10 males and 3/10 females. Minimal to mild mixed cellular inflammation was noted in 2/10 Mid dose females.
No test item-related findings were microscopically observed at a dose level of 10 mg/kg bw/day.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no test item related changes in the animal oestrous cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrous phases.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

pancreas

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 1: Mean Total Proten, Albumin and Albumin/Globulin ratio on Day 28 with Historical control data Males Dose group (mg/kg bw/day)   control 10 100 1000 Total protein (g/L) Mean 56.66 58.37 61.55** 62.46** Historical control range Min-Max. (mean) 49.60 – 63.90 (56.02) Albumin (g/L) Mean 29.99 31.91** 33.72** 34.45** Historical control range Min-Max. (mean) 26.50 – 35.40 (29.96) Albumin/Globulin ration Mean 1.12 1.21* 1.23** 1.24** Historical control range Min-Max. (mean) 0.90 – 1.40 (1.16) * = p<0.05, ** = p< 0.01 (Duncan’s Multiple Range test)

Table 2: Mean liver weights on Day 28
	Dose groups (mg/kg bw/day)
	control	10	100	1000
Males
Absolute (g)	13.75	13.68	13.97	14.88
	differences in %	-0.5	1.6	8.2
Body weight relative (%)	3.10	3.20	3.31*	3.39**
	differences in %	3.2	6.8	9.4
Brain relative(%)	642	622	659	710*
	differences in %	-3.0	2.6	10.6
Females
Absolute (g)	7.70	7.59	7.32	7.48
	differences in %	-1.4	-5.0	-2.9
Body weight relative (%)	3.10	3.23	3.08	3.15
	differences in %	4.2	-0.6	1.6
Brain relative(%)	384	389	370	382
	differences in %	-1.3	-3.6	-0.5
* = p<0.05, ** = p< 0.01 (Duncan’s Multiple Range test)

Table 3: Mean kidney weights on Day 28
	Dose groups (mg/kg bw/day)
	control	10	100	1000
Males
Absolute (g)	3.07	3.16	3.26	3.34
	differences in %	2.9	6.2	8.8
Body weight relative (%)	0.69	0.74*	0.77**	0.76**
	differences in %	7.2	11.6	10.1
Brain relative(%)	143	144	154	159*
	differences in %	0.7	7.7	11.2
Females
Absolute (g)	1.80	1.74	1.75	1.78
	differences in %	-3.3	-2.8	-1.1
Body weight relative (%)	0.73	0.74	0.74	0.75
	differences in %	1.4	1.4	2.7
Brain relative(%)	90	89	88	91
	differences in %	-1.1	-2.2	1.1
* = p<0.05, ** = p< 0.01 (Duncan’s Multiple Range test)

Conclusions:

The study was performed under GLP conditions and according to OECD 410. Due to the dose-dependent effects found in the exocrine tissue of the pancreas at 100 and 1000 mg/kg bw/day in males and females, a dose level of 10 mg/kg bw/day was considered to be the overall NOAEL for both males and females. No skin irritation was observed up to the highest dose tested, therefore a NOAEL local of 1000 mg/kg bw/day (equivalent to 8 and 5.16 mg/cm²/day for males and females, respectively).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5.16 mg/cm²

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises one adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in the respective endpoint summary.

Additional information

Data on repeated dose toxicity are available based on a series of studies following repeated exposure via the oral route over subacute, subchronic and chronic periods. In addition, the effects after subacute repeated exposure via the dermal route were investigated in the rat.

 

Oral

Subacute toxicity

Two subacute oral repeated dose toxicity studies performed in rats and mice are available. As the treatment duration lasted only 14 days, this is considered to be supporting data only. A short summary is provided below:

Oral 14-day repeated dose toxicity study in rats (M-202170-01-1):

Five Wistar rats/sex were treated by gavage with 0, 30, 150 and 600 mg/kg bw/day of test substance for 14 days. A satellite subgroup of three rats per sex was added to the high dose and control groups for interim sacrifice after 3 days of treatment to check only liver weight and hepatic cellular proliferation and histopathology. The following parameters in the main group were evaluated: detailed clinical observation, body weight, food consumption, ophthalmoscope examination, haematology, and clinical chemistry including cytochrome P-450 content, brain, kidney, liver, ovaries, spleen, testes and thyroid weights, gross pathology and histopathology of liver, kidney, spleen, ovaries, testes and thyroid gland. In the 600 mg/kg bw/day dose group only, higher liver weights and slight centrilobular hepatocellular hypertrophy in males were observed. Hepatocellular hypertrophy in combination with higher liver weights and without further histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. Therefore, the NOAEL was determined to be 600 mg/kg bw/day, which was the highest dose tested.

Oral 14-day repeated dose toxicity study in mouse (M-203945-01-1):

Five C57BL/6 J@Ico mice/sex were treated by gavage with 0, 100, 300 and 1000 mg/kg bw/day of test substance for 14 days. In the 1000 mg/kg bw/day dose group only, higher liver weights and centrilobular hepatocellular hypertrophy in males and females were observed. Hepatocellular hypertrophy in combination with higher liver weights and without further histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. Therefore, the NOAEL was determined to be 1000 mg/kg bw/day, which is the highest dose tested.

Subchronic toxicity

Five subchronic oral repeated dose toxicity studies are available; these comprise of 90-day repeated dose toxicity studies performed in rat, mouse and dog, respectively, and one neurotoxicity study performed in rats (this study is included within the Neurotoxicity section).

Oral 90-day repeated dose toxicity study in rats (M-232957-01-1):

The key study was considered to be the oral repeated dose toxicity study in rats, conducted according to OECD TG 408 (2003). The test substance was administered continuously via the diet to separate groups of Wistar rats (10/sex/group) at concentrations of 1.25, 600, 4000 and 12000 ppm (equivalent to approximately 0.08, 39.0, 259 and 787 mg/kg bw/day in males and 0.09, 45.6, 302 and 902 mg/kg bw/day in females, respectively) for at least 90 days. A similarly constituted group of 10 males and 10 females received untreated diet and acted as a control.

Clinical signs were recorded daily, body weight and food consumption were measured weekly. A detailed physical examination was performed once during the acclimatization phase and weekly throughout the study. In addition, grasping, righting, corneal, pupillary, auditory startle and head shaking reflexes were examined once during the acclimatization phase and during Week 12. Ophthalmological examinations were performed on all animals during the acclimatization phase and during Weeks 3, 8 and 12. The week before necropsy a blood sample was collected from the retro-orbital venous plexus of each animal for haematology and clinical chemistry determinations. Urine samples were collected overnight before necropsy from all animals. All animals were necropsied, selected organs weighed (from animals surviving to terminal sacrifice) and a range of tissues were taken, fixed and examined microscopically.

12000 ppm:

There were two male mortalities at 12000 ppm. One male was found dead on study Day 84, clinical signs prior to death consisted of pale eyes, cold to touch, general pallor, reduced motor activity and red soiled fur. A second male died during anaesthesia for blood sampling on study Day 86. This animal had lost weight during the previous week together with a corresponding reduction in food consumption. At necropsy, this animal had red soiling around the muzzle and a pale appearance. Treatment-related clinical signs in animals surviving to terminal kill consisted of white area on the eye of one male, anogenital soiling in one male and five females, hair loss in six females and wasted appearance in one female. Absolute body weight was reduced by between 9-12% in males and 6-10% in females during the whole treatment period, compared with the control groups (significant for males). The body weight gain was significantly reduced by 47% in males and 82% in females, after 1 week of treatment, with an overall reduction after 90 days of 19% (p>0.05) in males and 13% (p>0.05) in females, compared with the control group. Overall food consumption was significantly reduced by 6% in males, with the most marked effect during Week 1 when there was a 16% reduction compared with controls. In females the food consumption was reduced during Week 1 only, by 24% compared with the control females. Body weight changes are not considered to be adverse since they are significantly reduced over the whole period and are also in correlation with lower food consumption. At the ophthalmic examination, one male had a bilateral 'snow flake' corneal opacity together with neovascularisation of the cornea at Weeks 8 and 12, plus unilateral haemorrhaging in the iris at Week 3. A second male had unilateral haemorrhaging of the retina and bilateral pale retinal fundus at Week 12. The haematology evaluation showed a 13% increase (non-significant) in the prothrombin time in males, compared with control males. In addition, the male which died during anaesthesia for blood sampling had a very low erythrocyte count and haemoglobin concentration, together with a high leucocyte and neutrophil count, plus the presence of immature cells. At the clinical chemistry assessment, total cholesterol concentration was significantly increased by 45% in males and 34% in females, triglycerides concentrations were significantly increased by 81% in males and inorganic phosphorus concentration was significantly increased by 16% in females, compared with the relevant control groups. These effects show no clear dose dependency or relation to histopathological findings and are therefore not considered to be adverse. Urinalysis revealed a significant higher urinary volume in females. Marginally lower pH values and slightly higher ketone levels in both sexes are not considered to be an adverse effect since no clear dose response effect was observed. Absolute liver weight and relative weights were increased by between 22-38% in the high dose males compared with the control group. Microscopic examination of the two decedent males revealed similar treatment-related changes within the liver, pancreas and thymus. Both animals had a diffuse centrilobular acute hepatocellular necrosis in the liver, moderate acute/subacute pancreatitis with necrosis in the pancreas and moderate to marked single cell necrosis, mild to moderate atrophy/involution and slight multifocal parenchymal haemorrhages in the thymus. Microscopic examination of animals surviving to termination revealed treatment-related changes in the liver and pancreas of 6/8 surviving males. These changes consisted of slight to mild diffuse hepatocellular hypertrophy, mainly in the centrilobular region of the liver. Without any critical changes found in liver enzymes and the related liver increase this is a common adaptive response of the liver in rats found after treatment therefore, this effects is not considered to adverse. In the pancreas, 4/8 males had pancreatic lesion of which one was a mild acute/subacute pancreatitis with necrosis, similar to the lesion observed in decedent males. Other lesions included slight to mild atypical periductular fibrosis, interstitial oedema and the presence of interstitial intracytoplasmic golden brown pigmentation. These lesions correlate with the findings in the combined chronic and carcinogenicity study in rats (M-270092-01-1), where the pancreas was identified as a target organ, specifically adverse effects were observed in the pancreatic exocrine tissue (acinar atrophy/fibrosis).

Taken together male animals show adverse treatment-related effects when fed with 12000 ppm test substance. The effects found in females are not considered adverse since none died, and there was no correlation between the effects found in clinical chemistry and in histopathologic findings.

4000 ppm:

Treatment-related clinical signs consisted of white area on the eye in 2 males and anogenital soiling in 1 female. In females, absolute body weight was reduced by between 3-8%, with a reduction in body weight gain of 47% after 1 week of treatment and an overall reduction in body weight gain of 10% after 90 days, compared with the control group. There was a corresponding reduction in food consumption of 12% during the first week of treatment in females, compared with the control group. Therefore, this effect is not considered to be adverse. At the ophthalmic examination two males and one female had a unilateral 'snow flake' corneal opacity and neovascularisation of the cornea, which were present at Weeks 3, 8 and 12 in one male, Weeks 8 and 12 in the second male and Week 12 in the female. In addition, one female had unilateral haemorrhaging in the iris at Week 3. At the haematology evaluation the prothrombin time was found to be increased by 25% in the males which is considered to be in single occurrence and thus not adverse. Clinical chemistry assessment revealed a 47% increase in total cholesterol concentration and a 46% increase in triglyceride concentrations in males, compared with the control males. The effects were not clearly dose-related and for the triglycerides only a few animals were affected in each group. Therefore, these effects are not considered to be adverse. Urine analysis revealed marginally lower pH values and slightly higher ketone levels in both sexes compared with the control groups; these are not considered to be adverse because no clear dose response was observed. Absolute liver weight and relative weight were increased by between 30-41% in males, compared with the control males. At the microscopic examination, a slight to moderate diffuse hepatocellular hypertrophy, mainly in the centrilobular region, was observed in 10/10 males. Hepatocellular hypertrophy in combination with higher liver weights and without further histologic or clinical pathology alterations indicative of liver toxicity is considered to be an adaptive and a non-adverse reaction. An interstitial oedema within the pancreas associated with an interstitial mixed cell infiltrate was observed in 2/10 males. This is considered to be an isolated occurrence.

600 ppm:

One male and one female had a unilateral "snow flake" corneal opacity together with neovascularisation of the cornea at Weeks 8 and 12 in the male and Week 12 in the female, at the ophthalmic examination. This finding corresponded to the clinical sign of white area on the eye observed in these two animals. Total cholesterol concentration and triglyceride concentrations were increased by 43% (p<0.01) and 41% (p>0.05) respectively, in males, compared with the control group. These effects are not considered to be adverse because no corresponding histological changes were observed. Urine analysis revealed marginally lower pH values and slightly higher ketone levels in both sexes which are not considered adverse since no dose response was observed. Absolute and relative liver weights were increased by between 25-32% in males, compared with the control group. Microscopically, a slight to moderate diffuse centrilobular hepatocellular hypertrophy, mainly in the centrilobular region, was observed in 4/10 males. This is considered to be an adaptive response to the metabolic load of the test substance on the liver.

1.25 ppm:

No treatment-related findings were observed at 1.25 ppm in either sex.

In conclusion, the No Observed Adverse Effect Level (NOAEL) in the male Wistar rats when administered the test substance in the diet over a 90-day period was 4000 ppm (equivalent to 259 mg/kg bw/day) as in the 12000 ppm dose group two animals died and surviving animals showed histopathologic effects in the exocrine regions of the pancreas. For female rats the NOAEL was set to 12000 ppm (equivalent to 902 mg/kg bw/day) since no adverse effects were observed at the highest dose level tested. The ocular findings observed in the study are not considered to be relevant to humans, as discussed below under the section “Supplementary studies on the mode of action analysis and assessment of human relevance”.

Oral 90-day repeated dose toxicity study in mice (M-233114-01-1):

This study is considered to be a supporting study since it was performed similar to OECD TG 408 and in the mouse. The test substance was administered continuously via the dietary administration to groups of C57BL/6 J@ Ico mice (10/sex/group) at concentrations of 700, 2500 and 7000 ppm (equivalent to approximately 115, 411, and 1251 mg/kg bw/day in males and 150, 559, and 1564 mg/kg bw/day in females, respectively) for at least 90 days. A similarly constituted group of 10 males and 10 females received untreated diet and acted as a control.

Clinical signs were recorded daily, body weights and food consumption were measured weekly. On the day of necropsy, a blood sample was collected from the retro-orbital venous plexus of each animal for selected clinical chemistry determinations. All animals were necropsied, selected organs weighed and a range of tissues were taken, fixed and examined microscopically.

The test substance dietary administration for 90 days in C57BL/6 mice at dose level of 7000, 2500 and 700 ppm induced no treatment-related mortality.

7000 ppm

Treatment-related clinical signs consisted of coloured urine (intense yellow) observed from Day 6 to the end of the study in both sexes. A slight body weight loss (up to 4%) was observed during the first week of treatment in males and during the first two weeks in females of the 7000 ppm group. The rest of the treatment period, body weight changes were similar to the control values. As a consequence of the initial body weight loss, mean body weight was reduced throughout the study when compared to control values (up to -5% in males and -8% in females). The difference was statistically significant for half of the recorded days throughout the treatment period. Food consumption was unaffected by treatment. As the reduction was less than 10%, these effects are considered to be not adverse. The clinical chemistry evaluation revealed a statistically significant decrease in mean total cholesterol concentration in males (-33%) and in mean alkaline phosphatase activity in females (-28%), when compared to control values. Since these effects were the only significant clinical chemistry changes observed and no correlated histopathological effects were seen, these findings were not considered adverse. At terminal sacrifice, mean terminal body weight was significantly lower in males (-6%) when compared to control values. Mean absolute and relative liver weights were significantly higher in females (+29 and +22%, respectively), when compared to control values. The histopathological examination revealed treatment-related changes in the liver: a slight centrilobular hepatocellular hypertrophy associated with a slight tendency toward a decrease in incidence and severity of the centrilobular hepatocellular vacuolation was observed in both sexes. These liver effects are considered as an adaptive response to the treatment and are therefore treatment-related, but not adverse effects.

2500 and 700 ppm

Treatment-related clinical signs consisted of coloured urine (intense yellow) observed from Day 6 to the end of the study in both sexes. No other treatment-related effects were observed. This effect is not considered to be adverse as it does not have any clinical importance.

In conclusion, since all found effects are not considered to be adverse the No Observed Adverse Effect Level (NOAEL) of the test substance is 7000 ppm in males and females (approximately 1251 and 1564 mg/kg bw/day, respectively).

Oral 90-day repeated dose toxicity study in dogs; initial study (M-398594-01-2):

The study was performed in accordance to OECD TG 409 and under GLP. The test substance was orally administered to dogs for a period of 90 days (13 weeks) to evaluate subchronic toxicity of the test substance and determine a no-observed-adverse-effect level (NOAEL). Groups of 4 male and 4 female Beagle dogs were administered the test substance by incorporating it into the basal diet at a level of 0, 2000, 5000, or 12000/8000 ppm (at 12000 ppm until Week 2 and then at 8000 ppm).This was equivalent to 0, 57, 143 and 256 mg/kg bw/day in males and 0, 63.1, 153 and 265 mg/kg bw/day in females.

General and detailed clinical signs and mortality were observed, and body weights and food consumption were recorded for all animals during the treatment period. Ophthalmology, urinalysis, and haematological and blood biochemical examinations were performed on all animals prior to initiation of treatment and at Weeks 7 and/or 13. After termination of 13-week treatment, animals were euthanatized and subjected to organ weight measurements, necropsy, and histopathological examination. Treatment-related changes are summarized below.

12000/8000 ppm:

One female dog showed staggering gait and decreased spontaneous motor activity from Weeks 2 and 3, respectively, and red sebum at Week 6, and died at Week 7. Histopathologically, this animal showed foreign body pneumonia which was considered to be the cause of the death, but the relationship between the death and the treatment with the test substance was unknown. Three males and three females including this animal showed decreased spontaneous activity, staggering gait, and crouching position from Week 1 as demonstrated by general clinical observation. They showed decreased spontaneous motor activity and abnormal gait in the home cage and open field from Week 2 as recorded by detailed clinical observation; there were significant differences in scores of decreased spontaneous motor activity for males and abnormal gait for both sexes. Furthermore, there was a deterioration in systemic condition with evidence of hypothermia, loose stools, red sebum, salivation, dry nose, and vomiting of feed at Week 2.

Therefore, the dose level was changed from 12000 ppm to 8000 ppm at Week 3. However, significant clinical observations were still recorded at Weeks 3 and 4, and were persistently observed until week 8. Decreased body weight gain was observed in both sexes throughout the treatment period. There were statistically significant differences in mean body weights in males from Week 11 to the termination of treatment when compared with the control group. Ophthalmology revealed corneal opacity in one male and two females. Urinalysis exhibited significant increases in ketones and bilirubin, and a decrease or tendency to decrease in urinary pH in both sexes. At haematology, both sexes showed significant decreases in haematocrit (Ht), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC), and a significant increase or tendency to increase in platelet count (PLT). Both sexes also showed tendencies to increase in total leukocyte count (WBC) and neutrophil count, though there were no significant differences. Bone marrow cytology exhibited a significant increase in proerythroblast count (Proe) in both sexes, and a significant decrease and tendency to decrease in myeloid/erythroid ratio (M/E ratio) in males and females, respectively. Blood biochemistry revealed a tendency to increase in total bilirubin (T.Bil) in both sexes, though there was no significant difference.

Organ weight measurement revealed significant increases in relative weight of the liver and in absolute and relative weights of the spleen in both sexes, in absolute and relative weights of the kidney in males, and in absolute weight of the liver in females. At necropsy, dark-coloured spleen was found in three males and one female and enlarged spleen was found in two males. Histopathologically, increased haematopoiesis in the systemic bone marrow (rib, sternum, and femur) was observed in all males (4 of 4 animals) and all females (3 of 3 animals) at terminal kill, and their incidences were significantly increased when compared with the control groups (0 of 4 animals). Since the female that died at Week 7 showed increased haematopoiesis, the incidence of this finding was significantly increased in all females (4 of 4 animals) when compared with the control group (0 of 4 animals). In the dead female, increased haematopoiesis was comprised of particularly increased myelogenic cells, which might be caused by severe pneumonia, in contrast to increased haematopoiesis with increased erythroblasts in the other animals. In the spleen, congestion and increased deposition of brown pigments including hemosiderin were found in all males (4 of 4 animals) and all females (3 of 3 animals) at terminal kill, and their incidences were significantly increased when compared with the control groups (0 of 4 animals). Diffuse hepatocellular hypertrophy in the liver was observed in all females (3 of 3 animals) at terminal kill, and the incidence was significantly increased when compared with the control group (0 of 4 animals). Degeneration of corneal epithelial cells was observed in three males and three females including the dead female, some of which showed cornel opacity at ophthalmology. Axonal degeneration in the optic nerve was observed in three males and one female at terminal kill.

5000 ppm:

Two males and one female showed staggering gait and decreased spontaneous motor activity, both of which were noted at almost the same periods as described in the 12000/8000 ppm group. The female also showed salivation at Weeks 3 and 4. By detailed clinical observation, the female and one male showed decreased spontaneous motor activity and abnormal gait in the home cage and open field, and these signs were observed at the same periods together with staggering gait and decreased spontaneous motor activity. Decreased body weight gain was recorded in both sexes throughout the treatment period. Ophthalmology revealed corneal opacity in one male.

At urinalysis, a significant increase in ketones was detected in males, and a positive reaction to ketones was detected in all females. Low pH was noted in some animals of both sexes. At haematology, both sexes showed significant increases in WBC (particularly an increase in neutrophil count) and PLT, and significant decreases in MCV, MCH, Hb, and MCHC. Females showed a significant decrease in Ht. Bone marrow cytology revealed a significant increase in Proe and a tendency to decrease in M/E ratio in females. Organ weight measurement exhibited significant increases in relative weights of the liver and kidney in males, and a significant increase in absolute weight and a tendency to increase in relative weight of the liver in females. At necropsy, dark-coloured spleen was found in two males. Histopathologically, congestion in the spleen was found in all males and females (4 of 4 animals of both sexes), and their incidences were significantly increased when compared with the control groups (0 of 4 animals). Increased haematopoiesis in the systemic bone marrow was observed in all males (4 of 4 animals, with a significant difference) and three females at terminal kill. Degeneration of corneal epithelial cells was observed in three males and two females, and axonal degeneration in the optic nerve was observed in two males and one female.

2000 ppm:

One male showed decreased spontaneous activity at Weeks 8 and 9, and decreased spontaneous motor activity in the home cage and open field at Week 8. By general clinical observation, opacity in the eye was observed in one female. Decreased body weight gain was recorded in both sexes throughout the treatment period. Ophthalmology revealed corneal opacity in one male and two females. At urinalysis, a positive reaction to ketones was detected in all males and three females, and low pH was detected in both sexes. At haematology, both sexes showed a significant increase or tendency to increase in WBC (particularly an increase in neutrophil count), and a significant decrease in MCV; males showed a significant increase in PLT; females showed significant decreases in MCH, Hb, and MCHC. Organ weight measurement revealed a significant increase in relative weight of the liver in both sexes and a significant increase in absolute weight of the liver in females, and a significant increase in relative weight of the kidney in males. At necropsy, opacity in the eye was found in one female. Histopathologically, degeneration of corneal epithelial cells was observed in three males and three females, and increased haematopoiesis in the systemic bone marrow was observed in three males.

Based on the results mentioned above, the test substance-treated animals showed temporal appearance of clinical signs, decreased body weight gain, microcystic hypochromic anaemia, increases in platelet and leukocyte counts (particularly increased neutrophil count), and effects on the liver, kidney, spleen and eye. It was concluded that the no observed adverse effect level (NOAEL) of the test substance to Beagle dogs under the condition of the present study was less than 2000 ppm in both sexes (57.0 mg/kg bw/day in males and 63.1 mg/kg bw/day in females).

Oral 90-day repeated dose toxicity study in dogs; second study (M-398722-01-2):

The study was performed in accordance to OECD TG 409 and under GLP. Groups of four male and four female Beagle dogs were each given the test substance for a period of 90 days (13 weeks) by incorporating it into the basal diet at a level of 20, 200, or 2000 ppm (equivalent to 0.564, 5.72 and 58.6 mg/kg bw/day in males and 0.591, 5.57 and 62.1 mg/kg bw/day in females). A similarly constituted group of four males and four females received untreated diet and acted as a control.

Clinical signs, mortality, body weights and food consumption were recorded for all animals during the treatment period. Ophthalmological examinations were performed on all animals prior to initiation of treatment (Week 0) and at Week 13. Urinalysis, and haematological and blood biochemical examinations were performed on all animals prior to initiation of treatment (Week 0) and at Weeks 7 and 13. At termination of treatment, all surviving animals were euthanatized and necropsied, and organ weights were recorded. Histopathological examination was performed on all animals.

2000 ppm

No mortality or clinical findings were observed. Three males and three females showed treatment related body weight loss during the treatment period, with a range of 0.3 to 1.6 kg. Urinalysis revealed significant decreases in pH and a significant increase of ketone in males at Week 7. Low pH was observed across both sexes of all the treatment groups at Week 13. In addition, ketone-positive reactions were observed in both sexes at Weeks 7 and 13. These were considered to be effects of the test substance, as low pH and ketone-positive reaction were observed in both sexes and correlated with the findings in a preceding 90-day oral toxicity study (M-398594-01-2). Haematological examinations revealed significant decreases of the mean corpuscular volume and mean corpuscular haemoglobin in both sexes, and mean corpuscular haemoglobin concentration in males. Although these animals showed no alternations in haematocrit and haemoglobin, males showed a significant increase in erythrocyte count. However, in the preceding-90-day oral toxicity study, decreases in erythrocyte indices, haematocrit and haemoglobin were noted in the animals of both sexes at higher dose levels than those of this study, suggesting that there was microcytic hypochromic anaemia. Thus, they were considered to be a treatment-related effect on blood in this study as well. In addition, increases in platelet count and neutrophil count in females were also considered treatment-related, since these changes corresponded with the results of the preceding 90-day oral toxicity study. A significant shortening of activated partial thromboplastin time in males was considered to be incidental, since this was not observed in the animals at higher dose levels than those of this study in the preceding 90-day oral toxicity study. No significant effects on clinical chemistry parameters were observed. A significant increase in relative liver weight was observed in females, however, in the absence corresponding clinical chemistry and histopathological findings, this effect is considered adaptive rather than adverse. Females showed a significant decrease in absolute brain weight, however, there were neither effects on relative brain weight nor histopathological changes in the brain. In addition, there were no effects on brain weight in the preceding 90-day oral toxicity study. Thus, this change was considered incidental with no relevance to treatment of the test substance. Histopathologically, degeneration of corneal epithelial cells was observed in the eyes of three males and two females. This corresponded to the ophthalmological examinations, which revealed opacity of the cornea in two males and one female. In addition, histopathology showed increased haematopoiesis in systemic bone marrow (rib, sternum, and femur) in three males and two females. The increased haematopoiesis in the bone marrow in this study was also considered to be an effect of the test substance treatment, since a treatment-related effect on blood was observed in these animals, and the increased haematopoiesis in the bone marrow was also observed in all treatment groups in the preceding 90-day oral study.

200 ppm

No mortality or clinical findings were observed. Urinalysis revealed a significant decrease in urine pH in males at Week 7. Low urine pH was also observed in one male and two females at Weeks 7 and 13. No significant alterations in haematological parameters were observed in either sex. Blood biochemical examination revealed significant elevations of alkaline phosphatase in males at Week 7. These findings were considered incidental as they were not seen at the higher dose level, nor were they seen at higher dose levels in the preceding 90-day oral study. No effects on organ weight were observed at this dose level. Histopathologically, degeneration of corneal epithelial cells was observed in the eyes of one male and one female. Ophthalmology revealed opacity of the cornea in one female.

20 ppm

No mortality or clinical findings were observed. Urinalysis revealed low urine pH in two females at Week 7 and one male and two females at Week 13, although these changes were not significant in either sex. Males showed significant increases in eosinophil count at Weeks 7 and 13, however, these were considered incidental since these findings were not observed at the higher dose levels. Blood biochemical examination revealed significant elevations of alkaline phosphatase in males at Week 7. These findings were considered incidental as they were not seen at the higher dose level, nor where they seen at higher dose levels in the preceding 90-day oral study. No effects on organ weight were observed at this dose level. Histopathologically, degeneration of corneal epithelial cells was observed in the eyes of one male and three females. Ophthalmology revealed opacity of the cornea in two females, one of whom also had vascularization of the cornea.

Triketone-type compounds constitutively similar to the test substance inhibit 4-hydroxyphenylpyruvate dioxygenase (4-HPPDase) which is an important enzyme of the tyrosine metabolic pathway, leading to increased concentrations of tyrosine in the blood. When rats are administered 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexandione (NTBC) which is a triketone showing a typical inhibitory activity toward 4-HPPDase, corneal lesion occurs after the increase of tyrosine in the anterior aqueous humor along with the increase of tyrosine in the blood. This lesion can be induced by tyrosine crystals being taken into lysosome in corneal epithelial cells, leading to degeneration and necrosis in corneal epithelial cells and subsequent inflammation in those cells in rats. This common mode of action of HPPDase inhibitors with regards to ocular toxicity is discussed by the US EPA in a review paper of HPPDase inhibiting herbicides (M-808741-01-1). Therefore, it is considered that degeneration of corneal epithelial cells which was observed in both sexes in all the treatment groups was due to test substance related increases in the concentrations of tyrosine in the blood. Ocular lesions considered to be the result of the same mechanism were also observed in male and female rats in the chronic toxicity study conducted with the test substance (M-270092-01-1). The ocular lesion in the rat was diagnosed as keratitis, as reported in several literatures, since severe infiltration of inflammatory cells was observed in addition to degeneration and necrosis of corneal epithelial cells. However, infiltration of inflammatory cells was not observed in any animals with the corneal lesion, although vascularization of the cornea was observed in one female dog at 20 ppm.

In conclusion, the No Observed Adverse Effect Level (NOAEL) of the test substance in Beagle dogs when administered in the diet over a 90-day period was less than 20 ppm (equivalent to 0.564 and 0.591 mg/kg bw/day in males and females, respectively), as ophthalmological findings, characterized by degeneration of corneal epithelial cells and corneal opacity was seen in all dose levels. These observations are considered not to be relevant to humans, as discussed below under the section “Supplementary studies on the mode of action analysis and assessment of human relevance”.

Chronic

Combined chronic toxicity (24-month) and carcinogenicity study in the rat (M-270092-01-1):

The oncogenic potential and the chronic toxicity of the test substance were assessed in a study performed according to OECD TG 453 and in compliance with GLP. Wistar Rj:WI (IOPS HAN) rats were fed a diet containing 0, 2, 50, 1500 and 5000 ppm of the test substance for at least 104 weeks. Seventy-five rats per sex were used in the control and high dose groups (10 animals/sex designated for the interim sacrifice after 52 weeks, 15 animals/sex for the recovery group sacrifice after 68 weeks and 50 animals/sex for the final sacrifice after 104 weeks). Sixty rats per sex were used in the intermediate groups (10 animals/sex for the interim sacrifice and 50 animals/sex for the final sacrifice). Observations and examinations included mortality and clinical signs, detailed physical examinations including palpation for masses, body weight, food consumption, ophthalmological examinations, test substance analysis in the blood, haematology, clinical chemistry determinations and urinalysis. At the scheduled chronic, recovery and carcinogenicity phase sacrifice, selected organs were weighed and designated tissues sampled and examined microscopically. The mean achieved dose levels of the test substance received by the animals over the 12-month period were approximately 0.09, 2.33, 72.0 and 245 mg/kg bw/day in males and 0.13, 3.21, 99.6 and 337 mg/kg bw/day in females. The mean achieved dose levels of test substance received by the animals over the 24-month period were approximately 0.08, 2.03, 62.4 and 214 mg/kg bw/day in males and 0.11, 2.83, 88.6 and 296 mg/kg bw/day.

Over the first 12 months of the study, the mortality rate was low (1-5 per dose group) at most dose levels for both sexes with no evidence of a treatment or dose-related increase. In the male high dose group 16/75 animals were found dead or were sacrificed prematurely for humane reasons, principally within the first 6 weeks of the study. The main clinical signs in the early decedent males consisted of ocular or nasal discharge, soiled nose, soiled fur or anogenital region, wasted appearance, together with the usual signs associated with morbidity. Macroscopic examination indicated a haemorrhagic lesion as the cause of premature death in at least eight of these males. During the second year of the study, no treatment-related effect was noted on the mortality incidence. After 2 years, 14-40 animals per dose group of 50 animals were found dead or sacrificed prematurely.

5000 ppm:

Treatment-related clinical signs observed in a significant number of animals consisted of an increased incidence of white area on eyes (neovascularization as found in ophthalmologic examination, see below), localized soiled fur, soiled anogenital region, hair loss, reduced motor activity, cold appearance, general pallor, wasted appearance, and dental abnormality during the first year of the study in males and/or females. During the second year, the treatment-related clinical signs observed in a significant number of animals were: white area on eyes, skin lesions, reduced motor activity and focal swelling (principally of hindlimb) in males, and of white area on eyes, chromodacryorrhea and soiled anogenital region in females. At the end of the recovery phase, all clinical signs were found to be reversible, with the exception of white area on eyes that was observed in a single male only, and were therefore not considered to be adverse effects.

During the first week of treatment, mean body weight was lower by 5 and 4% and mean body weight gain by 21 and 29%, in males and females, respectively, compared to controls. This trend was observed throughout the course of the study, leading to a final body weight reduction on Day 708 of 5 and 15% in males and females, respectively, and to a body weight gain reduced by 6 and 23% in males and females, respectively, when compared to controls. No relevant changes in body weight were noted during the recovery phase; indicating that these effects were reversible. The food consumption was slightly reduced by up to 6% during the first week of treatment in both sexes, but was similar to controls thereafter. No significant changes in food consumption were noted during the recovery phase. The ophthalmological examinations revealed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. After 13 weeks of recovery, the corneal opacity, oedema of the cornea and snow flake-like corneal opacity were completely reversible, whereas neovascularization of the cornea was still observed in some high dose animals from both sexes. The type of corneal lesions observed at 5000, 1500 and 50 ppm in the rat (together with the eye keratitis noted at the microscopic examination), are characteristics of a compound such as the test substance that inhibits 4-hydroxyphenylpyruvic acid dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. These lesions are related to an increase in plasma tyrosine level caused by a blockade of the 4-HPPDase enzyme in the rat. However, the rat is a species particularly sensitive to inhibition of the 4-HPPDase enzyme and is atypical in its susceptibility to develop tyrosine-related eye lesions. Therefore, although these lesions (corneal lesions and eye keratitis) were treatment-related, they were considered not to be toxicologically relevant to humans. This is discussed further under the section “Supplementary studies on the mode of action analysis and assessment of human relevance”.

The clinical chemistry assessments showed a higher mean total cholesterol concentration up to Month 18 of the study in both sexes, together with a higher mean triglycerides concentration up to Month 12 in males and up to Month 3 in females, in comparison to controls. All changes noted were reversible after the 3-month recovery period. In the absence of clear changes in the histopathological examination, they were considered to be treatment-related, but not adverse, effects.

Urinalysis showed higher ketone level throughout the study in both sexes, and lower pH values during the first year in both sexes and during the second year in males, when compared to controls. In addition, higher protein level and a lower amount of crystals were observed in males up to Month 18, compared with controls. After 13 weeks of recovery, only the tendency towards a lower pH value was observed in females, while the rest of the above-mentioned changes were reversible. The observed effects on urinary parameters were considered not to be adverse, due to the reversibility and lack of corresponding histopathological effects.

At the end of the chronic phase (12 months) the mean absolute and/or relative kidney weights were higher in males at 5000 ppm (statistically significant) in comparison to controls. These changes were considered to be treatment-related.  Mean absolute and relative liver weights were higher in males at 5000 ppm (statistically significant), in comparison to controls. These changes were considered to be treatment-related. However, in view of the limited changes noted at the microscopic observation, they were considered to have no toxicological relevance. Mean absolute brain weight was decreased by 6% (p<0.05) in males at 5000 ppm, in comparison to controls. This change was considered not to be toxicologically relevant, since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations. All organ weight changes detected at the end of the chronic phase were found to be reversible. The few changes noted after the 3-month recovery period were considered to be incidental in view of their individual variation.

At the end of the carcinogenicity phase (24 months), the mean absolute and relative liver weights were increased by 25 and to 31%, respectively, in males, while the relative liver weight was increased by 17% in females. These changes were considered to be a treatment-related, adaptive response to the test substance exposure. Mean absolute and relative kidney weights were increased by 16 and 22%, respectively, in males, in comparison to controls. During the macroscopic examination, toxicologically relevant changes were noted in the kidneys. The incidence of irregular surface on kidney was increased in females (3), in comparison to controls (0). In the kidney, the incidence of minimal to severe chronic progressive nephropathy was statistically significantly higher in males (38), compared with controls (30). However, this change is a common age-related and spontaneously occurring disease in the rat and was observed with a high incidence in control males (30/50). This change is not considered to be toxicologically relevant. The histopathological examination showed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, effects were observed in the exocrine tissue, with the incidence of acinar atrophy/fibrosis higher in both sexes (36 for males, 31 for females vs. 18 for males, 13 for females in control). In the thyroid gland, the incidence of follicular cell hypertrophy was elevated in both sexes (27 for males, 19 for females vs. 3 for males, 1 for females in control). Other changes noted during the histopathological examination were considered not to be toxicologically relevant.

1500 ppm

The treatment-related clinical signs consisted of a significantly increased incidence of white area on eyes in both sexes, together with soiled anogenital region in 4/60 females during the first year. During the 2nd year, a significantly increased number of males had white area on eyes, reduced motor activity and focal swelling (principally of hindlimb), compared with controls; while an increased number of females had a white area on eyes, compared with control.

The mean body weight was reduced by up to 6% during the first year of treatment in both sexes, in comparison to controls, while the mean body weight gain was reduced by up to 11 and 18% throughout the first year in males and females, respectively. The effect persisted in both sexes, as the final body weight on Day 708 was reduced by 9 and 15% in males and females, respectively, when compared to controls. The body weight gain was reduced by 14 and 21% in males and females, respectively, when compared to controls. The food consumption was unaffected by treatment.

The ophthalmological examinations revealed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. These observations are considered not to be relevant to humans, as described above.

At the end of the chronic phase (12 months) the clinical chemistry evaluation showed higher mean total cholesterol and/or triglycerides concentrations were noted in both sexes (statistically significant on most occasions), in comparison to controls. Lower mean alkaline phosphatase activity was noted in males at Months 3 and 6 (statistically significant). These changes were considered to be treatment-related. However, since the decrease was observed only transiently and in the absence of any changes at the histopathological examination, they were considered not to be toxicologically relevant. Slight variations were also observed in males in total protein and albumin concentrations and consequently in globulin concentrations and albumin/globulin ratio (calculated parameters), principally at Month 3. However, the magnitude of the variations relative to the controls was less pronounced throughout the sampling periods. Therefore, these differences were considered to be meaningless and not to be toxicologically relevant. After the carcinogenicity phase no changes were observed.

The urinalysis showed higher ketone level on most occasions during the study in both sexes, compared with control. Lower mean pH values compared to controls were observed in both sexes throughout the first year and only in males throughout the second year, though no clear dose related effect was noted in males, a lower amount of crystals was observed up to Month 18, together with a higher protein level at Months 6, 12 and 18.

At the end of the chronic phase (12 months), the mean absolute and relative liver weights were higher in males (statistically significant only for the relative liver weight), in comparison to controls. These changes were considered to be treatment-related. However, in view of the limited changes noted at the microscopic observation, they were considered to have no toxicological relevance. Mean absolute and/or relative kidney weights were higher in males (statistically significant for the relative weight parameter), in comparison to controls. These changes were considered to be treatment-related. The mean absolute brain weight was decreased by 7% (p<0.01) in males at 1500 ppm, in comparison to controls. This change was considered not to be toxicologically relevant, since it was observed for this parameter only and was not associated with any change at the macroscopic and microscopic examinations.

At the end of the carcinogenicity phase (24 months), the mean absolute and relative liver weights were higher in males (25% and 36%, respectively, p<0.01), when compared to controls. Mean relative weight was also higher in females (12%, p<0.05), in comparison to controls. There were no toxicologically relevant findings during the macroscopic examination. The histopathological examination showed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, effects were observed in the exocrine tissue, with the incidence of acinar atrophy/fibrosis higher in both sexes (33 in males and 32 in females vs. 18 in males and 13 in females in controls). In the thyroid gland, the incidence of follicular cell hypertrophy was elevated in both sexes (19 in males and 20 in females vs. 3 in males and 1 in females in controls). Other changes noted during the histopathological examination were considered not to be toxicologically relevant.

50 ppm

Treatment-related clinical signs were limited to a significantly increased incidence of white area on eyes in both sexes throughout the study, together with a significant increase in the incidence of reduced motor activity and focal swelling (principally of hindlimb) in males during the second year. The Body weight parameters were comparable to control values in both sexes during the first year of treatment.

Throughout the second year, the mean body weight was reduced by up to 11 and 8% and the mean body weight gain by up to 17 and 12% in males and females, respectively, compared to controls. The food consumption was unaffected by treatment.

The ophthalmological examinations showed a high incidence of corneal opacity, neovascularization and oedema of the cornea and snow flake-like corneal opacity in both sexes throughout the study. These observations are considered not to be relevant to humans, as described above.

The clinical chemistry evaluation showed a significantly higher mean total cholesterol level up to Month 12 of the study in males and a significantly higher mean triglycerides concentration on one or two sampling periods in both sexes, in comparison to controls.

The urinalysis showed higher ketone level on most occasions during the study in both sexes and lower pH values during the first year in both sexes and also during the second year in males, when compared to controls. In addition in males, a lower amount of crystals was observed up to Month 18, together with a higher protein level at Months 6, 12 and 18.

At the end of the chronic phase (12 months), the mean kidney weights were increased by between 17 to 22% in males, in comparison to controls. There were no toxicologically relevant findings during the macroscopic examination. The few changes noted during the histopathological examination were considered to be incidental.

At the end of the carcinogenicity phase (24 months), the mean relative liver and kidney weights were significantly increased by 20% each in males, when compared to controls. There were no toxicologically relevant findings during the macroscopic examination. The histopathological examination revealed toxicologically relevant changes in the pancreas and thyroid gland, in comparison to controls. In the pancreas, effects were observed in the exocrine tissue, with the incidence of acinar atrophy/fibrosis higher in females (28 vs. 13 in controls). In the thyroid gland, the incidence of follicular cell hypertrophy was elevated in both sexes (16 in males and 22 in females vs. 3 in males and 1 in females in controls). Other changes noted during the histopathological examination were considered to be incidental.

2 ppm

No toxicologically relevant changes were noted throughout the course of the study in either sex for any of the parameters evaluated.

In conclusion, due to adverse effects on the exocrine tissue of the pancreas and the thyroid gland in males and females, the NOAEL over a 24-month period of dietary administration with the test substance was 2 ppm (equivalent to 0.08 and 0.11 mg/kg bw/day in males and females, respectively). The histopathological changes observed in thyroid, such as follicular cell hypertrophy resulted from excessive metabolism of thyroid hormones by phase II conjugation enzymes induced by the treatment with the test substance, as demonstrated in study M-400191-01-2 (for details of this study, refer to section “Supplementary studies on the mode of action analysis and assessment of human relevance”).

1-year repeated dose toxicity study in dogs (M-398593-01-2):

The study was performed in accordance to OECD TG 452 and under GLP. Groups of four male and four female Beagle dogs were each given the test substance for a period of 52 weeks by incorporating it into the basal diet at a level of 1, 4, 20 or 2000 ppm (equivalent to 0.0247, 0.102, 0.515 and 53.5 mg/kg bw/day in males and 0.0255, 0.102, 0.514 and 53.6 mg/kg bw/day in females). A similarly constituted group of four males and four females received untreated diet and acted as a control.

During the treatment period, all animals were checked for general and detailed clinical conditions and mortality. In addition, their body weights and food consumption were recorded. Animals were also subjected to ophthalmology, urinalysis, haematology, and blood biochemistry before initiation of treatment and at Weeks 13, 26 and 52. After 52 weeks of treatment, all animals were euthanized and subjected to organ weight measurement, necropsy, and histopathology.

There were no deaths in any treatment groups during the treatment period. Treatment-related changes observed in each dose group are summarized below.

2000 ppm:

Ophthalmology revealed corneal opacity in two males and one female. Among these dogs, two males showed opacity of the eye during the general and detailed clinical observations, and at necropsy. Histopathology showed degeneration of corneal epithelial cells in each of the three dogs. Abnormalities of the faeces was seen in females (soft, mucosal or bloody mucosal faeces). Males and females showed a depression of body weight gain. Urinalysis revealed several males and females who were positive for ketones, and low urine pH was observed in females. Haematology revealed significant decreases in haematocrit, haemoglobin concentration, mean corpuscular volume and mean corpuscular haemoglobin in males and females. Significant increases in platelet count in males and females, and erythrocyte count in females were also noted. Clinical biochemistry revealed significant increase in alkaline phosphatase in males. Organ weight measurement revealed significant increases in absolute and relative kidney weights in males.

20 ppm:

Ophthalmology revealed corneal opacity in one male and two females. Among these dogs, one male and female showed opacity of the eye during the clinical observations. Urinalysis revealed several males and females who were positive for ketones, additionally, low urine pH was observed in some females.

4 and 1 ppm

There were no treatment-related changes that were considered adverse.

As with the previously conducted 90-day repeated dose toxicity studies in dogs (M-398594-01-2 and M-398722-01-2), the ophthalmological findings are concluded to be caused by a rise in blood tyrosine concentration due to inhibition of 4-hydroxyphenylpyruvate dioxygenase (4-HPPDase) by the test substance, which is an important enzyme in the tyrosine metabolic pathway in mammals. When blood tyrosine concentrations are elevated, it results in high tyrosine concentration in the anterior chamber fluid of the eye. Subsequently, tyrosine crystal uptake into lysosomes of the corneal cells occurs, leading to corneal lesions. Further mechanistic information on corneal effects induced by the test substance is discussed below under “Supplementary studies on the mode of action analysis and assessment of human relevance”.

In conclusion, the No Observed Adverse Effect Level (NOAEL) of the test substance in Beagle dogs when administered in the diet over a 52-week period was 4 ppm (equivalent to 0.102 mg/kg bw/day in males and females), as in the 20 and 2000 ppm groups, adverse effects were noted on the eye and urinary properties, and in the 2000 ppm group depression of body weight gain and adverse effects on blood, liver and kidneys were noted.

Taking together all results from oral repeated dose toxicity studies, the chronic toxicity study in rat had the lowest NOAEL, based on the adverse effects observed in the pancreas and thyroid. Therefore, the NOAEL of 0.08 mg/kg bw/day found in male and female rats, is used for classification and risk assessment.

Dermal, subacute exposure

Dermal 28-day repeated dose toxicity study in rats (M-447472-02-1):

A 28-day dermal toxicity study according to OECD TG 410 and under GLP was performed with the test substance in rats. Each experimental group consisted of 10 Wistar rats/sex. The test substance was applied to the rat skin (shaved flank) and covered with a semi-occluded application at doses of 10, 100 and 1000 mg/kg bw/day, for 6 h. The test substance was held in place using special jackets. The test substance was gently wiped from the skin with lukewarm water after the 6-hour exposure period to prevent ingestion. Control animals were similarly treated with gauze patches and restrainers (Lomir jackets with inserts).

Parameters monitored during the study were morbidity and mortality, clinical observations and neurological and ophthalmological examination. Local clinical signs were evaluated following the patch removal, monitoring to observe any signs of test item-related irritation according to the Draize score system. Body weights of each animal were recorded at randomization, on Day 0 and then weekly; food consumption were recorded on Day 0 and then at weekly. Clinical pathology evaluation (haematology, clinical chemistry and urinalysis) and examination of the vaginal smears were conducted prior to necropsy, performed on Day 28. Terminal body, brain and selected organ weights were measured. Full histopathology was performed in Groups 1 (Control) and 4 (High dose) animals. In addition, any organs or tissues with microscopic abnormalities were subjected to histological examination from all groups until a no effect level was established.

There was no unscheduled mortality during the study, and no clinical signs or skin irritation related to test substance administration were observed. No test substance-related effects were noted during the ophthalmoscopic examination or during the functional observation battery tests, landing food splay test or grip strength evaluation. There were no test substance-related or adverse effects on the animal body weights, body weight gain or animal food consumption during the study as well as no adverse effects on of the test substance on the haematology or urinalysis. Evaluation of the vaginal smears prior to necropsy showed the expected distribution of the oestrous cycle phases within the normal population of female Wistar rats. Changes that were considered to be related to test substance treatment were noted in the clinical chemistry parameters. Albumin (Alb.) concentration was slightly higher than control in all male dose groups, reaching statistical significance (p<0.01). Consequently Total protein (Tot.Prot) and the Albumin to Globulin ratio (A/G) were also higher, reaching statistical significance (p<0.05 or p<0.01). However, there were no histopathological or other changes associated with these differences at any dose level. As all values in the 10 mg/kg bw/day group fell within the normal historical control range, these changes were considered to be non-adverse at this dose level. There were no test substance-related changes at dose levels of 10, 100 and 1000 mg/kg bw/day at necropsy.

Statistically significant differences in male liver (ca. 10%) weights at 1000 mg/kg bw/day were noted when adjusted to body weight or brain weight, which correlate with the histopathological findings described below. A consistent increase in absolute kidney weight, kidney weight relative to body weight and kidney weight relative to brain weight, was observed in High-dose males, compared with the corresponding control group. The magnitude of the increase was very similar for both the absolute and relative organ weights, ranging from 8.8 to 11.2%. This finding correlates well with the histopathological findings observed in the kidney at terminal sacrifice in the high dose males (as discussed in regulatory position paper M-477086-01-1). There were no biologically significant differences among groups in the weights of other organs measured, related to body weight or brain (compared to control). There was a slightly higher kidney weight in the Low- and Mid-dose males, but as the values were all within the laboratories historical control range and there was no indication from histopathology of any changes, the statistical difference was considered not to reflect a test item effect (as discussed in regulatory position paper M-478777-01-1). Test substance-related microscopic findings were noted in the liver, kidneys and pancreas. The microscopic findings in the liver and kidneys were in correlation with organ weight changes in the males. In the liver, the centrilobular hepatocellular hypertrophy was only observed in males dosed at 1000 mg/kg bw/day. Numerous findings were seen in the kidneys including increased presence of eosinophilic droplets in the cortical/corticomedullary tubules, tubular proteinaceous casts, tubular basophilia or dilatation of renal pelvis. The changes seen were considered to represent an increased incidence of common background features. However, the casts were accompanied with minimal tubular degeneration in one male at the High dose. These renal changes were ascribed to treatment with test substance only in High dose males. Casts were also present in one High dose female but the incidence and severity could be within the normal range, so the observation cannot be unequivocally ascribed to treatment. An additional histopathological evaluation of the animals dosed at 100 mg/kg bw/day did not show any microscopic changes in the females. In the males, tubular cast was only present in one rat without accompanied alterations; therefore this change was not ascribed to treatment with the test substance. There were no significant differences between treated males and females in treatment-related findings of the pancreas. Increased acinar zymogen granules, necrosis of acinar cells or mixed cell inflammation were microscopically visualized. The severity of these lesions was decreased when comparing 1000 mg/kg bw/day-group (minimal to moderate) versus 100 mg/kg bw/day-group (minimal to mild). Based on the proximity of these findings, the effects were considered to only be associated with the exocrine tissue of the pancreas. No test substance-related microscopic findings in the pancreas were recorded in rats dosed at 10 mg/kg bw/day.

Due to the dose-dependent effects found in exocrine tissue of the pancreas at 100 and 1000 mg/kg bw/day in males and females, a dose level of 10 mg/kg bw/day was considered to be the overall NOAEL for both males and females. No skin irritation was observed up to the highest dose tested, therefore a NOAEL local of 1000 mg/kg bw/day (equivalent to 8 and 5.16 mg/cm²/day for males and females, respectively, based on a generic modification recommended by REACH Guidance R.8, p.112 as follows: NOAELtest*mean weight/44.5 = NOAELmodified where 44.5cm² is 10% of an average total body surface of a rat) was considered.

Supplementary studies on the mode of action analysis and assessment of human relevance

In vitro inhibition assay to 4-hydroxyphenylpyruvate dioxygenase (4-HPPDase) (M-398583-01-2)

The test substance is categorized as a triketone-type herbicide and it is speculated that its mode of action depends on inhibiting the biosynthesis of plant chlorophyll based on the inhibition of 4-HPPDase, an enzyme of the tyrosine metabolic pathway. To determine the effect of the test substance on the inhibition of 4-HPPDase in mammals, the IC50 value, which inhibits 50% of enzymatic activity, of the test substance was measured in the hepatic enzyme system in rat (male Wistar Hannover GALAS).

An already-known positive control substance for 4-HPPDase inhibition, 2-(2-Nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione (NTBC) exhibited 100% of inhibition at the final concentration of 1E-6 mol/L in the pre-test. The results indicated that the method in this study is appropriate.

For the test substance, more than 40% inhibition to 4-HPPDase activity was obtained at concentrations of 1E-9 mol/L or higher and 100% inhibition was detected at a concentration of 1E-7 mol/L. The results strongly indicated that the test substance has a potential to inhibit 4-HPPDase activity. The inhibition rate was 13.8% at a concentration of 1E-11 mol/L, while it was 0.0% at a concentration of 1E-10 mol/L. No dose-dependency on these two concentrations was considered within an error of approximately 10%, according to the data obtained from the main test.

The IC50 value for the inhibition of 4-HPPDase activity induced by the test substance was 1.68E-9 mol/L, indicating that the test substance inhibits 4-HPPDase activity even at a low concentration, as noted in an already-known positive control substance for 4-HPPDase inhibition, NTBC.

Time course changes of the tyrosine-concentrations after single oral administration in the plasma of rats, mice and rabbits (M-398580-01-2)

After a single oral administration of the test substance in rats (Crj:CD (SD)), mice (Crlj: CD-1 (ICR)) and rabbits (Slc:JW/CSK) at dose levels of 1, 10, 100 and 1000 mg/kg bw (3 males/species/dose group), blood samples were collected at 2, 6, 24, 48 and 72 h. Using these samples, tyrosine concentrations in the plasma were determined, and possible species differences within the development of tyrosine concentrations in the plasma were defined. For comparison, NTBC was used as positive control and was orally administrated once at 10 mg/kg bw.

In rats, mice and rabbits, tyrosine concentrations in the plasma were elevated among all species at the dose levels of ≥ 1 mg/kg bw when compared to those of the vehicle control animals.

The duration to reach maximum concentrations showed a tendency of delay at higher dose levels, at 1000 mg/kg bw, maximum concentrations were nearly reached after 24 h.

When comparing the plasma-tyrosine concentrations to each other among all dose levels, maximum concentrations were nearly already achieved at 100 mg/kg bw for all species. The maximum tyrosine concentrations in the plasma were below 1000 µmol/L in the mice and rabbits, showing no striking species difference. In contrast to this, the maximum tyrosine concentrations in the plasma of the rats were greater than 2000 µmol/L, which was far beyond the concentrations in the mice and/or rabbits. When comparison was made in detail, the maximum tyrosine concentrations in the plasma of the mice and rabbits at 100 mg/kg bw were 633 µmol/L and 877 µmol/L, respectively, whereas the value in the rats was 2061 µmol/L at the same dose level, being 3.3 and 2.4 times higher as compared to mice and rabbits, respectively.

Similar results were achieved in the animals receiving NTBC, where the maximum tyrosine concentrations were below 1000 µmol/L in the mice and rabbits, therefore, showing no remarkable species difference. However, in the rats the maximum tyrosine concentration was greater than 2000 µmol/L, which was markedly higher than those in the mice and/or rabbits. That is, the maximum concentrations in the plasma of the mice and rabbits were 583 µmol/L and 534µmol/L, respectively, whereas it was 2590 µmol/L in the rats. This means that the value in the rats was 4.4 and 4.9 times greater than those in the mice and rabbits, respectively.

In summary, when a single dose of the test substance was given orally to rats, mice and rabbits, the tyrosine concentrations in the plasma were elevated at the dosages of ≥1 mg/kg bw in all species, and a definite interspecies difference was recognized in the maximum tyrosine concentrations of the plasma of animals receiving 100 and 1000 mg/kg bw.

Overtime assessment of changes in plasma tyrosine concentration after single administration in dogs (M-398581-01-2)

The study was conducted in order to assess overtime changes in plasma tyrosine concentration after single administration of the test substance at dose levels of 1, 10 and 100 mg/kg bw to Beagle dogs. Blood sampling and measurement of plasma tyrosine concentrations were conducted on three females at 0 (before administration), 3, 6, 9, 12, 24, 48, 72 and 168 h after single administration.

The test substance was administrated by using a gradual increase method at each dose level. That is, 21 day and 14 day intervals were placed after the blood sampling period (7 days) for the 1 mg/kg bw and 10 mg/kg bw dose groups, respectively, and then 10 and 100 mg/kg bw dosing was conducted. During the test period, observations of general clinical condition and body weight measurement were conducted. Plasma tyrosine concentrations were measured at each blood sampling point.

There were no treatment related effects to the clinical observations, body weight or food consumption.

In all dose levels, plasma tyrosine concentrations were increased from 3 h after administration and the maximum value of mean tyrosine concentrations became more than 1000 µmol/L at 10 and 100 mg/kg bw as shown in the table below. After reaching maximum levels, plasma tyrosine concentrations decreased rapidly and became almost equal to the values before administration, after 48 h at 1 mg/kg bw and 168 h at 10 and 100 mg/kg bw.

Maximum values of mean plasma tyrosine concentration:

Dose level (mg/kg bw)	Maximum concentration (µmol/L)	Time-to maximum plasma concentration (hours)
1	603	12
10	1038	24
100	1452	24

In conclusion, after single gavage administration of the test substance in Beagle dogs, plasma tyrosine concentrations rapidly increased and the maximum concentrations reached more than 1000 μmol/L at 10 and 100 mg/kg bw dose levels.

Overtime assessment of changes in plasma tyrosine concentrations after dietary administration in rats (M-398575-01-2)

Time course changes of tyrosine concentrations in the plasma of rats was evaluated in groups of Sprague Dawley rats (CRL: CD(SD)IGS) consisting of three males. Rats were fed the diet containing the test substance at concentrations of 0 (control), 1, 10, 100 and 1000 ppm over a period of 14 days (exposure period). These animals were then fed test substance-free diet for 7 days (recovery period). During both periods, concentrations of tyrosine in the plasma of rats was periodically analysed. In addition, general conditions, body weight, food consumption and chemical intake on these animals were monitored.

During the 14-day exposure period, one animal in the 100 and 1000 ppm groups showed whitish eyes, which was reversible during the recovery period.

During the experiment, there were no notable changes on body weight development of the animals. Although reduced food consumption in the 100 and 1000 ppm groups was temporarily recorded after the initiation of exposure, food consumption was stable thereafter. The mean daily chemical intake during the 14-day exposure period was 0.068 mg/kg bw/day at 1 ppm, 0.68 mg/kg bw/day at 10 ppm, 6.90 mg/kg bw/day at 100 ppm and 66.5 mg/kg bw/day at 1000 ppm.

At 1 ppm, the concentrations of tyrosine in the plasma were comparable with those of the control animals. However, in the animals at ≥ 10 ppm, the concentrations of tyrosine were markedly increased on Day 1 of the exposure and peaked on Day 3, specifically 1276 µmol/L at 10 ppm, 3040 µmol/L at 100 ppm and 3429 µmol/L at 1000 ppm. After that, although the concentrations of tyrosine in these animals showed a tendency for slight reduction, the concentrations were close to initial concentrations during the 14-day exposure period. The concentrations of tyrosine at 10 and 100 ppm were reduced to the level of the control animals the day after replacing the diet with a test substance-free one. At 1000 ppm, this reduction was seen on Day 3 of recovery. During the administration period, one rat in each of the 100 and 1000 ppm groups showed whitish eyes, however, this change disappeared during the recovery period.

In summary, when animals were a fed diet containing the test substance at dose levels of ≥10 ppm, the concentrations of tyrosine in the plasma were markedly increased, and high concentrations of tyrosine were relatively persistent during 14-day exposure period, although there was a tendency for slight reduction. The concentrations of tyrosine in the plasma were, however, quickly reduced and returned to normal levels after termination of exposure. The increased tyrosine level was accompanied by whitish eyes observed in the test animals exposed to 100 and 1000 ppm during the administration period.

Time course changes of the tyrosine-concentrations in the plasma of mice (M-398573-01-2)

Groups of mice (Crlj:CD-1 (ICR)) consisting of three males were fed a diet containing the test substance at the dietary concentrations of 0 (control), 0.5, 5, 50 and 500 ppm over the period of 14 days. During the exposure period, blood samples were periodically collected, and the tyrosine concentrations in the plasma were determined. In addition, after the feeding period with the test substance, mice received the basal diet for 7 days (recovery period), during which tyrosine concentrations in the plasma were also periodically monitored.

There were no treatment related changes in clinical observations, body weight or food consumption. The mean daily chemical intake during the 14-day exposure period was 0.0755 mg/kg bw/day at 0.5 ppm, 0.741 mg/kg bw/day at 5 ppm, 7.43 mg/kg bw/day at 50 ppm and 72.3 mg/kg bw/day at 500 ppm.

Time course changes of the tyrosine concentrations in the plasma:

Group	Tyrosine concentration in the plasma (µM)
	Administration period			Recovery period
	Day 1	Day 7	Days 10 - 14	Day 7
0 ppm	122	123	105 - 130	84
0.5 ppm	126	425*	253 - 366	97
5 ppm	629*	612	613 - 616	138
50 ppm	785*	761	781 - 677	202
500 ppm	663	793*	726 - 749	555

*: maximum

In the mice fed the diet containing the test substance, the concentrations of tyrosine in the plasma were elevated at all dose groups when compared to those of the control group. Peak levels of tyrosine were observed approximately 7 days after initiation of the exposure at 0.5ppm. In contrast to this, it required 1 – 3 days at the dose levels of ≥ 5 ppm, showing quick elevation in these groups. The maximum concentrations (mean value) in each group were 425, 629, 785 or 793 µmol/L at 0.5, 5, 50 or 500 ppm, respectively.

It was shown that the concentration at 50 ppm was comparable to that at 500 ppm, suggesting that saturation had possibly taken place already at 50 ppm. After reaching the peak concentrations, the concentrations of tyrosine were persistent and comparable to the maximum levels in general for each dose group during the rest of the exposure period, suggesting that tyrosine levels were held in equilibrium.

After the termination of exposure to the test substance, the concentrations of tyrosine were quickly reduced at 0.5, 5 and 50 ppm. Seven days after the termination of the exposure (Day 7 of the recovery period), the concentrations of tyrosine at 0.5 and 5 ppm were comparable to that of the control group, and at 50 ppm were reduced to slightly higher levels than that of the control group (84 µM vs 202 µM for control and 50 ppm animals). Although a tendency for a reduction of tyrosine was seen in the 500 ppm group, the rate of reduction was rather slow, and higher concentrations than that of the control still remained even on Day 7 of recovery (555 µM at 500 ppm compared to 84 µM in controls).

In summary, test substance caused an increase in tyrosine concentrations in the plasma of mice at dietary dose levels of ≥ 50 ppm. After termination of exposure, the concentration of tyrosine was quickly reduced at ≤ 50 ppm, although the reduction rate was delayed at 500 ppm.

In vitro evaluation of tyrosine metabolism in the Liverbeads™ from different species after inhibition of 4-HPPDase (M-281188-01-1)

The objective of this study was to evaluate the levels of tyrosine and hydroxyphenyl lactic acid (HPLA) in suspensions of rat, dog, rabbit, mouse and human Liverbeads™ following incubation with the test substance, in order to check and compare their ability to metabolize cellular tyrosine via a substitute enzymatic pathway producing the metabolite HPLA. The time periods of incubation were 0, 2 and 4 hours. These time points were chosen according to a preliminary validation study.

When the test substance was incubated alone with the Liverbeads™ from the different species, the analysis of HPLA levels displayed the following results:

HPLA was not detected in rat and dog Liverbeads™ at the 0 hours time point or was barely detected at very low traces (not quantifiable) at the 4 hour incubation time.

In the rabbit and human Liverbeads™, HPLA levels were also barely detected at 0 and 2 hours, but HPLA was detected at trace levels at the 4 hour incubation time.

In the mouse Liverbeads™, HPLA levels were 0.14µg/mg protein at 0 hours, and increased during the incubation periods (up to 0.49 µg/mg protein at the 4 hour incubation time).

The test substance was also incubated under tyrosine burden conditions, with Liverbeads™ from the same species as above, in order to determine the ability of each species to react under these tyrosine saturating conditions.

The analysis of HPLA levels displayed the following results:

HPLA was not detected at 0 hours in rat, dog and rabbit. Low traces (“<LOQ–trace”) of HPLA were detected in human, and only the mouse presented amounts of HPLA reaching two fold the basal level (0.27 µg/g protein).

At 2 hours, HPLA was present at very low traces (“<LOQ–trace”) in the dog Liverbeads™ or present at traces level in the rat Liverbeads™. Levels of HPLA were clearly measurable in human, rabbit and mouse with mean HPLA levels of 0.14 µg/g protein in the human, 0.28 µg/g protein in the rabbit and up to 0.72 µg/g protein in the mouse Liverbeads™.

At 4 hours, HPLA was still present at traces level in the dog and in the rat Liverbeads™. In the rabbit Liverbeads™, the level of HPLA appeared to be stable compared to the concentration measured at 2 hours. At the same incubation time, concentrations of HPLA had doubled in the mouse and human Liverbeads™ reaching respectively 1.25 µg/g protein and 0.32 µg/g protein.

These results showed that the species used in this study can be classified into 2 main groups:

1: rabbit, human and mouse which are able to produce HPLA and to use an alternative pathway for the tyrosine catabolism when HPPDase is inhibited.

2: dog and rat, for which this alternative pathway is much less efficient under “basal” and “extreme” conditions.

These results showed that rabbit, human and mouse had a high efficiency to use an alternative pathway mediated by 4-HPLA for the tyrosine catabolism when HPPDase was inhibited by the test substance.

Analysis of tyrosine, 4-HPPA and 4-HPLA in plasma and urine after single administration of the test substance to rat and mouse (M-399482-01-2)

It is considered that when a 4-HPPD (4-hydroxyphenylpyruvate dioxygenase) inhibitor is administered to animals, tyrosine, which is a precursor of 4-HPPA (4-hydroxyphenylpyruvate), increases as a result of the increase in 4-HPPA caused by the suppression of the metabolism of HPPA to HGA (homogentisic acid). In addition, there are interspecific differences in the accumulation of tyrosine and it is also considered that tyrosine is not accumulative in the blood of mice because mice metabolize tyrosine to 4-HPPA and 4-HPLA and excrete those metabolites into the urine more quickly than rats.

This study was performed to clarify if the interspecific difference in the accumulation of tyrosine observed after administration of the test substance, a 4-HPPD inhibitor, was caused by the excretion capacity of tyrosine via metabolisation. After the test substance was administered once orally to rats and mice at a dose level of 100 mg/kg bw, the concentrations of tyrosine in the plasma and the metabolites of tyrosine (4-HPPA and 4-HPLA) in the urine were analysed. Tyrosine and its metabolites were shown as amount (µg) per body weight (kg). Male Crl:CD (SD) IGS rats at 8 weeks old and male Crlj:CD1 (ICR) mice at 6 weeks old were used.

Time course changes of concentrations of tyrosine metabolites in the urine:

Animals	Dose	Concentrations of tyrosine metabolites in the urine (mg/kg bw)
		4-HPPA		4-HPLA
		0 - 8 hours	0 - 48 hours	0 - 8 hours	0 - 48 hours
Rat	Solvent control	0.016	<0.041	0.039	0.273
	100 mg/kg bw	2.05	44.0	0.739	23.7
Mouse	Solvent control	0.040	<0.229	0.101	0.597
	100 mg/kg bw	49.0	101	8.59	29.5

The results show that the maximum amount of tyrosine in the plasma of rats was 2.9-fold larger than those of mice. In contrast, the total amount of 4-HPPA and 4-HPLA excreted into the urine during 0 - 8 hours after administration was 24-fold and 12-fold larger in mice than in rats, respectively.

Mice showed faster elimination of tyrosine in the plasma and faster excretion of metabolites (4-HPPA and 4-HPLA) into the urine than rats. Mice also showed the tendency not to accumulate tyrosine at higher levels in the blood. These results showed that the interspecific difference in the accumulation of tyrosine observed after administration of the test substance was likely to be caused by the different excretion capacities of tyrosine via metabolisation.

Mechanistic study for hepatic drug-metabolizing enzyme induction in rats (M-400191-01-2)

The present study was conducted to further examine the effects of the test substance on the liver and thyroid which were observed in the previously conducted "Chronic toxicity and carcinogenicity study in the wistar rat by dietary administration (M-270092-01-1)". Male and female SPF Wistar Hannover (BrlHan:WIST@JCL[GALAS]) rats were fed test diets containing the test substance at 0, 50 and 5000 ppm for 14 days (equivalent to 0, 4.39 and 407 mg/kg bw/day and 0, 4.88 and 453 mg/kg bw/day in males and females, respectively).

During the treatment period, all animals were observed for general condition and mortality. Further, body weights and food consumption were monitored. After 7 and 14 days of the treatment, blood sampling and measurement of blood T3, T4 and TSH levels were conducted followed by organ weight measurement and necropsy. At necropsy, a part of the liver tissue was sampled from each animal and stored frozen. The activity of hepatic drug-metabolizing enzymes, including Cytochrom P-450 enzymes, two isozymes (ethoxycoumarin O-dealkylase, ECOD and pentoxyresorufin O-dealkylase, PROD), and UDP-glucuronosyl transferase (UDPGT) were measured against 4-nitrophenol and 4-hydroxybiphenyl as substrates using microsomal fractions prepared from the frozen liver samples. The treatment-related changes are summarized as follows:

5000 ppm group: Males and females showed a significantly low body weight when compared to those of the controls at Weeks 1 and 2. Food consumption in males and females at Week 1 was significantly lower. In the measurement of blood hormone levels, T3 and T4 in males after 1 and 2 weeks of treatment and T3 in females after 1 week of treatment were significantly decreased. No alteration on blood TSH levels was determined. In the organ weight measurement, a significant increase was noted in relative weight of the liver in males after 1 and 2 weeks of treatment. Females showed significant increases in absolute and relative weights of the thyroids after 1 week of treatment. Hepatic microsomal protein content was significantly increased after 1 and 2 weeks of treatment in males. A significant increase in cytochrome P-450 content was noted in males and females after 1 week of treatment and males after 2 weeks of treatment. Females showed significant increases in activities of ECOD and PROD after 1 and 2 weeks of treatment. In the measurement of hepatic UDPGT activity, males and females showed significant increases in UDPGT activities against both of 4-nitrophenol and 4-hydroxybiphenyl as substrates after 1 and 2 weeks of treatment.

50 ppm group: Females showed significantly low body weight when compared to the control after 1 and 2 weeks of treatment. In the measurement of blood hormone levels, T3 and T4 in males were significantly decreased at Weeks 1 and 2. Females showed a significant decrease in blood T3 level at Week 1, but not at Week 2. In the organ weight measurement, significant increases in relative weight of the liver after 1 and 2 weeks of treatment and absolute weight of the liver after 2 weeks of treatment were noted in males. Females showed significant increases in absolute and relative weights of the thyroids after 1 week of treatment. Measurement of cytochrome P-450 isozyme activity revealed significant increases in P-450 content in males and females after 1 week of treatment and PROD activity in females after 1 week of treatment. In measurement of hepatic UDPGT activity, a significant increase in UDPGT activity against 4-nitrophenol as a substrate in males and females, and 4-hydroxybiphenyl in males was noted after 1 and 2 weeks of treatment.

In summary, these results show that the test substance induced hepatic drug metabolizing enzymes in rats and that thyroid hormones were excessively metabolized. However, as blood TSH was not altered by treatment, the effect on the thyroid hormone brought by the induction of drug metabolizing enzymes was not strong enough to evoke proliferation of the thyroid follicular cell other than hypertrophy. Therefore, it was considered that histopathological changes observed in thyroid such as follicular cell hypertrophy at the end of the carcinogenicity evaluation phase of the combined chronic toxicity and carcinogenicity study resulted from excessive metabolism of thyroid hormones by phase II conjugation enzymes induced by the treatment with the test substance.

 

Additional supporting information relating to ocular effects:

Exploratory 14-day (ocular toxicity) study in the rat and mouse (M-210983-01-2)

Groups of five male and five female CD rats, Brown Norway rats and CD 1 mice were administered either basal diet or diets supplemented with 2 or 5% tyrosine for 14 days. Animals were weighed weekly, observed for clinical signs daily and subjected to ophthalmological examinations on Days 2, 3, 7, 8 and 14. At study termination, eyes were taken from selected animals for histological examination and plasma taken for free plasma tyrosine analysis of selected groups.

No treatment-related clinical signs were observed in CD rats of either sex at 2%, or in either sex of Brown Norway rats or CD 1 mice at 2 or 5% tyrosine.

The observations in the 5% tyrosine male CD rat group were restricted to dark urine (all males during the second week of study and 3/5 females on Day 14) and one male which appeared thin and showed ptosis and piloerection from Days 10 and 13, respectively. In this animal, corneal opacity was severe and visible to the naked eye.

Body weights and food consumption were not affected by treatment.

No treatment-related corneal effects were observed in female groups of rats at 2% or 5% tyrosine. Male CD and Brown Norway rats were not affected at 2% tyrosine. No mice of either sex were affected at any dose. Slight corneal opacities were noted for three of five male CD rats at 5% tyrosine on Day 2. On the following day, these opacities had progressed to "moderate" and a fourth animal in the group showed a "slight" opacity. By Day 7, the opacities in these four animals had become "severe" and the fifth male had developed a "slight" opacity. By Day 14, two males developed a very severe opacity and showed signs of oedema and vascularisation of the cornea. In three animals, congestion of the iris became evident. Only one Brown Norway rat at 5% tyrosine developed any corneal changes; in this animal the corneal opacity was slight and was only observed on Day 14.

The corneal lesion similar in all animals was characterized by focal or multi focal areas of opacity, each lesion having a "snow-flake" appearance. In the most advanced case the whole cornea become involved, with evidence of oedema and vascularisation at Day 14.

Mean plasma tyrosine levels per group:

Groups	Strain	Dietary free tyrosine (%)	Mean Plasma Tyrosine (mg/L)
			Males		Females
			Mean	SD	Mean	SD
1	CD rat	0	21	4.2	13	2.0
2		2	59	4.8	-	-
3		5	114	39.9	62	29.8
4	Brown Norway (BN) rat	0	12	0.6	-	-
5		2	32	15.0	-	-
6		5	68*	13.0	-	-
7	CD 1 mouse	0	13	1.8	-	-
9		5	18	7.1	-	-

- Not determined

*excluding one high outlier

In male CD rats, the 2% and 5% tyrosine diets caused a 3-fold increase in plasma tyrosine to 59 mg/L and a 5-fold increase to 114mg/L. Only the 5% group developed corneal opacity. In females, the basal levels were lower than in males, but the percentage increase in plasma tyrosine was similar in the 5% group for males and females. In male Brown Norway rats, the basal plasma tyrosine levels were similar to female CD rats, as were the levels with 5% diets (68mg/kg). The levels for the 2% and 5% dietary male groups were about three fold and five fold the basal level, respectively. One male in the 5% group had a very high plasma tyrosine level (nearly 10 times that of the other four rats in the group), and was the only Brown Norway rat with a corneal lesion.

In CD 1 mice, the basal levels were similar to female CD rats and male Brown Norway rats but with a 5% tyrosine diet, there was no significant increase in plasma tyrosine levels.

At necropsy, no significant gross changes attributable to treatment were observed except for the corneal opacities described below.

Following microscopic examination, no treatment-related changes were seen in female CD rats at any dose level or in males at 2% tyrosine. The two male rats examined at 5% tyrosine presented qualitatively and quantitatively similar lesions. They were characterized by the following changes:

- diffuse corneal epithelial intracytoplasmic vacuolation

- severe interstitial oedema of the basal epithelial layer of the cornea

- oedematous and/or swollen nuclear changes in the corneal epithelial cells

- inflammatory reaction involving the whole cornea: diffuse polymorphonuclear (PMNs) cell infiltration of the corneal stroma and epithelium, focal infiltration of the corneal epithelium and ciliary processes.

These changes were correlated to the corneal opacities (superficial keratitis) observed for almost two weeks at the ophthalmologic examinations.

No treatment-related changes were seen in male Brown Norway rats at 2% tyrosine. One of the examined eyes from the 5% male group was from the only animal in the group of five to show changes visible at ophthalmoscopy. The histological changes in this eye were characterized by:

- focal accumulation of PMNs in the anterior chamber

- mild diffuse infiltration of the corneal stroma by PMNs lining up at the base of the epithelium

- solitary focus of superficial epithelial desquamation of non-keratinized cells in central portion of cornea.

These changes were correlated with the observation of a slight corneal opacity which was first detected shortly before necropsy.

No treatment-related changes were observed for any group of male mice.

In summary, addition of 5% tyrosine to the diet of male CD rats resulted in the rapid onset (48 hours) of corneal opacities with a "snow-flake" appearance (a superficial keratitis). At study termination, corneal lesions were correlated with elevated plasma tyrosine levels. One of five Brown Norway male rats at 5% tyrosine was found to have slight bilateral opacities at 14 days and a high plasma tyrosine level. No corneal opacities were seen in any group given 2% tyrosine diets or in any female rats, or in mice of either sex.

Overall discussion on corneal effects

In the available repeat dose toxicological studies, the liver was a target organ of the test substance in the rat, mouse and dog. Further, the eye was a target organ in both the rat and dog and the kidney in the rat and mouse. In addition, in the rat, the pancreas and thyroid were affected, whilst in the dog the haemopoietic system was affected resulting in the dogs showing signs of anaemia.

The test substance did not show any neurotoxicity, carcinogenicity, teratogenicity or genotoxicity potential and no adverse effects on reproduction or development were observed.

Effects on the eyes and liver, especially, the changes in the eyes were characteristic of a 4-Hydroxyphenylpyruvate dioxygenase (4-HPPDase) inhibitor (M-808741-01-1). While changes in the eyes such as keratitis and degeneration of corneal epithelial cells were observed in rats and dogs, mice did not show changes in the eyes. The underlying mechanism of these changes were considered as follows: It is known that triketone-type compounds constitutively similar to the test substance inhibit 4-HPPDase in the liver which subsequently leads to an increase in the concentration of tyrosine in the blood. After the increase of tyrosine in the anterior aqueous humour along with the increase of tyrosine in the blood, tyrosine crystal is taken into lysosome in corneal epithelial cells. This leads to degeneration, necrosis and inflammation in corneal epithelial cells. It is also known that there is an interspecific difference in the ability to metabolize tyrosine, which is also discussed by the ECETOC (ECETOC TECHNICAL REPORT No. 99, 2006) and US EPA (M-808741-01-1). It was shown that the test substance inhibited 4-HPPDase activity in rat hepatic enzyme preparation and increased the concentration of tyrosine in the plasma and the concentrations exceeded 1000 µM in rats and dogs, where eye lesions were observed, but did not exceed 1000 µM in mice and rabbits. In the study using cultured hepatocytes from rats, rabbits, dogs, mice and humans, it was determined that the ability to produce 4-HPLA was higher in rabbits, mice and humans than in dogs and rats. In addition, in the study where the concentration of tyrosine metabolites in the urine was compared between rats and mice, mice excreted tyrosine metabolites (4-HPPA and 4-HPLA) into the urine more rapidly than rats and mice had a tendency to accumulate less tyrosine than rats in the plasma.

Based on these results, it is considered that the test substance shows similar effects as triketone-type 4-HPPDase inhibitors, i.e., in mice, rabbits and humans, as urinary tyrosine excretion rates to use an alternative pathway mediated by 4-HPLA were higher than in rats and dogs, tyrosine was not accumulated in the plasma above a certain level, therefore, the possibility to cause the eye lesions in humans is extremely low.

Overall, as also discussed by the ECETOC (ECETOC TECHNICAL REPORT No. 99: Toxicological Modes of Action: Relevance for Human Risk Assessment (2006)) and US EPA (M-808741-01-1), corneal effects induced by 4-HPPDase inhibitors represent species-specific ocular toxicity in rats caused by high concentrations of tyrosine at the target side and not a direct action of the xenobiotic. Further, the experimental data on tyrosine plasma changes provide sufficiently conclusive evidence that tyrosine levels in dogs rapidly increase after treatment with the test substance, similar to rats. Thus, corneal effects observed in the dog are likewise considered as species-specific ocular toxicity caused by the action of high tyrosine levels. Based on the fact that HPPDase inhibition in man will not lead to tyrosine concentrations high enough to induce ocular toxicity, the observed corneal effect is not considered as relevant for humans. An extrapolation of corneal toxicity observed exclusively in rats and dogs to humans is considered as inappropriate. 

 

Justification for classification or non-classification

The available data for repeated dose toxicity with 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione (CAS 473278-76-1) meets the criteria for classification according to Regulation (EC) No 1272/2008, and has therefore to be classified as STOT RE Cat. 1 with the pancreas (exocrine) and thyroid gland as target organs.