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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21 Sep1998

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-chloro-4-methanesulfonyl-3-{[(oxolan-2-yl)methoxy]methyl}benzoyl)cyclohexane-1,3-dione

EC Number:

695-022-6

Cas Number:

473278-76-1

Molecular formula:

C20 H23 Cl O7 S

IUPAC Name:

2-(2-chloro-4-methanesulfonyl-3-{[(oxolan-2-yl)methoxy]methyl}benzoyl)cyclohexane-1,3-dione

Test material form:

solid

Test animals

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

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.25 ppm

Remarks:

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

Doses / concentrations 2

Dose / conc.:

600 ppm

Remarks:

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

Doses / concentrations 3

Dose / conc.:

4 000 ppm

Remarks:

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

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

3

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

Target system / organ toxicity

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

Any other information on results incl. tables

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	-	-	-

Applicant's summary and conclusion

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.