2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 July 2001 to 4 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.4300

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF, 2000 (Combined Chronic Toxicity/Oncogenicity Study)

Deviations:

yes

Remarks:

- 10 rats/sex were used for the 12-month chronic toxicity from all dose groups; reticulocyte counts were not analysed; gamma glutamyl transferase and triglyceride levels were not conducted and A/G ratios were not calculated.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

EC Number:

604-721-7

Cas Number:

150114-71-9

Molecular formula:

C6H4Cl2N2O2

IUPAC Name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: tan powder

Specific details on test material used for the study:

Purity: 94.5%

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks
- Weight on study day 1: 149.4 g for males and 113.4 g for females
- Housing: During acclimation animals were housed two-three per cage in stainless steel cages. During the main study, animals were housed two per cage in stainless steel cages. Cages had wire mesh floors and were suspended above catch pans. Cages contained a feed container and pressure activated nipple-type watering systems.
- Diet: ad libitum
- Water: ad libitum (municipal water)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.4 - 24.7 °C
- Humidity: 40-69 % (relative)
- Air changes: 12-15 changes per hour
- Photoperiod: 12 hour light/dark photocycle

IN-LIFE DATES
- From: Test material administration began on the 14 August 2001 for the males and the 15 August 2001 for the females
- To: Necropsies took place on the 13 August 2002 for the males and the 14 August 2002 for the females

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diet mixes were fed to the animals for a 2-week period before a new diet mix was prepared.
- Mixing appropriate amounts with (Type of food): 7 % premix was mixed with the diet to form the test diets.
- Storage temperature of food: Ambient temperature in sealed vessels.

Diets were prepared by serially diluting a concentrated test material- feed mixture (premix) with ground feed. Premixes were mixed periodically throughout the study based on stability data. Initial concentrations of test material in the diet were calculated from pre-exposure body weights and feed consumption data. Subsequently, the concentrations of the test material in the feed were adjusted weekly for the first 13 weeks of the study and at four-week intervals thereafter, based upon the most recent body weight and feed consumption data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the premixes and all the mixes, including the control diet, were performed prior to dosing and at approximately 4, 8 and 12 months of the study. The test material was extracted with solvent and analysed using liquid chromatography-mass spectrometry (LC-MS).
The homogeneity of the mixtures was determined in the low dose diet for the females and the high dose male test diet. The analyses were performed prior to dosing and then again at around 4, 8 and 12 months. Additional homogeneity determinations were performed when the pre-mix concentration was changed from 3 to 7 %.
Data on stability was available from a previous four-week study performed in mice which demonstrated that the test material was stable for at least 21 days in rodent chow. An additional stability test for the 7 % premix study was conducted up to 55 days.
One reference sample per sex per dose per mix were retained and stored at ambient temperature in sealed vials.

RESULTS
The concentrations of the test material were determined for the control and test diets from nine time points and were found to be acceptable. The mean concentrations for each dose level over the course of the study ranged from 96.9 to 105 % of targeted concentration.
No test material was found in the control diet. LC-MS analysis of each individual sample indicated 78.4-121 % of the target concentration, with the exception of one value of 135 %. A follow-up analysis using the same diet mixing instructions resulted in 90.7 % of the target concentration.
The homogeneity in rodent feed was determined for nine separate mixing batches for the 5 mg/kg/day female and 1000 mg/kg/day male test diets. In addition, homogeneity was conducted on the 50 and 1000 mg/kg/day female diet mixes and the 7 % premix and 1000 mg/kg/day female diet mix. The homogeneity of the diets was considered acceptable, with relative standard deviations for all diets sampled between 2.33 and 15.9 %, with the exception of one analysis where the relative standard deviation was 43.71 % for a sample from the 5 mg/kg/day female dose group. The relative standard deviation for the majority of samples was < 10 %.
Stability of the test material was determined in the 7 % premix and was determined to be stable in the premix for at least 55 days, at which it was 96.5 % of the concentration found initially.

Duration of treatment / exposure:

12 months

Frequency of treatment:

Daily; continuous in the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The high dose was selected based on preliminary results of a subchronic dietary toxicity study with a four week recovery period. The cecal weights were around 2.8 times the control values for males and females dosed with 1000 mg/kg/day for 13 weeks. Males had very slight epithelial hyperplasia of the cecum and ileum. The high dose selected was therefore expected to produce increased weight of the cecum and a decreased urine pH. The other doses were expected to provide a dose response relationship for any effect that might be observed at the highest dose tested for this study. The low dose was expected to produce a NOEL.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for mortality and moribundity at least twice daily. Other observations were carried out at least once a day.
- Cage side observations: Cage side observations included but were not limited to decreased/increased activity, repetitive behaviour, vocalisation, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency and faecal/urinary quantity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre-exposure then monthly until termination

BODY WEIGHT: Yes
- Time schedule for examinations: Pre-exposure, weekly for the first 13 weeks and then monthly thereafter.

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

FOOD EFFICIENCY:
- Calculated as (g/feed consumed/day)/( g bodyweight gain): Yes (first 13 weeks of the study)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure then at termination using indirect ophthalmoscopy
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6 and 12 months
- Anaesthetic used for blood collection: Yes, CO₂
- Animals fasted: Yes
- How many animals: 10 rats/sex/dose
- Parameters checked: Haematocrit, haemoglobin concentration, red blood cell count, total white blood cell count, platelet count, differential white blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3, 6 and 12 months
- Anaesthetic used for blood collection: Yes, CO₂
- Animals fasted: Yes
- How many animals: 10 rats/sex/dose
- Parameters checked: Prothrombin time, alkaline phosphatase activity, alanine aminotransferase activity, aspartate aminotransferase activity, albumin, cholesterol, creatinine, electrolytes (Ca, Na, K, phosphate and Cl), glucose, total bilirubin, total protein and urea nitrogen.

URINALYSIS: Yes
- Time schedule for collection of urine: 3, 6 and 12 months
- Metabolism cages used for collection of urine: Yes (overnight, 16 hours). Urine was also collected by manual compression of the urinary bladder.
- Animals fasted: No
- Parameters checked: colour, appearance, specific gravity, urine volume, pH, bilirubin, glucose, proteins, ketones, blood and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No (addressed in a separate group)

Sacrifice and pathology:

NECROPSY
Fasted rodents submitted alive for necropsy were anaesthetised by the inhalation of CO₂. The tracheas were exposed and clamped and the animals were euthanised by decapitation.
A complete necropsy was conducted on all animals and included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened glass slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined.
All visceral tissues were dissected from the carcass, re-examined, and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10 % formalin.
The brain, cecum (full and empty), liver, kidneys, heart, adrenals, testes, epididymides, ovaries, uterus, and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.

GROSS PATHOLOGY: Yes. Representative samples of tissues listed in Table 1 were collected and preserved in neutral, phosphate-buffered 10 % formalin.
HISTOPATHOLOGY: Yes. The number of sections from all preserved tissues listed in Table 1 were processed by standard histologic procedures from control- and high-dose group animals and all animals that died or were sacrificed in a moribund condition. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with haematoxylin and eosin and examined using a light microscope. The following tissues from the remaining groups were processed and histopathologically examined: liver, kidneys, lungs, cecum, spleen and all relevant gross lesions. The target organ (cecum) was microscopically examined from the low- and intermediate-dose group animals to define a NOEL.

Statistics:

Bodyweights, feed consumption, organ weights, urine volume and specific gravity, clinical chemistry, coagulation and haematologic data were evaluated using Bartlett’s test for equality of variances (α=0.01). Parametric or non-parametric analysis of variance (ANOVA) (α=0.05) were performed based on these results. A Dunnett’s test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons was performed (α=0.05) if significant.
Clinical observations were analysed by a z-test of proportion (α = 0.05). Descriptive statistics were reported for bodyweight gains, feed efficiency, RBC indices and differential WBC counts. Statistical outliers were identified by a sequential test (α = 0.02) but only excluded from feed consumption and feed efficiency statistics.
Statistical analyses were conducted on bodyweight, feed consumption, haematologic parameters, clinical chemistry parameters, urine volume, urine specific gravity and organ weight data throughout the study (geriatric changes were accounted for). Cumulative histopathologic incidences for all animals were used in the statistical analyses.
For tissues where all animals were examined, specific histopathological incidences were evaluated for deviation from linearity (α=0.01) using ordinal spacing of the doses. Linear data were tested with the Cochran-Armitage Trend test. If statistically significant (α=0.02) or significant deviation from linearity was observed, incidences were compared to the control using a pair-wise Chi-square test with Yates’ continuity correction (α = 0.05, two sided). For tissues evaluated from all control and high dose rats, but not all rats from intermediate doses, statistical analysis consisted of pairwise comparisons of the control and high dose group. Rare tumours (background incidence ≤1%) were considered significant in the Chi-square test with Yates’ continuity correction (α=0.10, two sided). Gehan-Wilcoxon procedure was used to evaluate the differences in mortality.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower for males and females at 1000 mg/kg and lower for males at 500 mg/kg

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Increased in males at 1000 mg/kg/day

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

AST levels were increased in 1000 mg/kg females at 3 and 6 months; the toxicological significance of this could not be determined.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Consistent changes for both sexes at 500 and 1000 mg/kg (increased volume, decreased specific gravity, pH, protein and ketones)

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

500 and 1000 mg/kg/day males and females had increased absolute and relative full and empty cecal weights

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged cecum at 500 and 1000 mg/kg/day

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Very slight, diffuse hyperplasia of the mucosal epithelium of the cecum of most rats given 1000 mg/kg/day and three males in the 500 mg/kg/day group.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All tumours observed were considered to be incidental.

Details on results:

CLINICAL SIGNS AND MORTALITY
No statistically significant differences in mortality rates for males and females at any dose group.
There were no statistically significant or treatment related changes in clinical or detailed clinical observations in any treated group. Periocular soiling was found in one-two animals per sex per dose, however this was found to be sporadic and not attributed to treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
Bodyweights for males dosed with 500 and 1000 mg/kg/day were lower than controls. The difference between treated animals and control developed gradually during the study and reached statistical significance at day 120 where the males at 500 and 1000 mg/kg/day weighed 2.4 and 3.8 % less than controls, respectively. These groups were generally statistically identified for the remainder of the study. Males at 50 mg/kg/day had slightly lower bodyweights than controls, however this was not attributed to treatment as they were within 97 % of the control bodyweights and were only transiently statistically identified. Males at 5 mg/kg/day had slightly lower bodyweights, but this was not statistically identified at any point. The initial bodyweights of male rats dosed with 5 or 50 mg/kg/day were slightly lower than the rats in the other dosing groups.
Bodyweights for females dosed with 1000 mg/kg/day were slightly lower than the controls and remained so throughout the study. These were considered to be treatment related. The decrement developed gradually and was maintained at approximately the same level throughout the study. This was statistically identified at day 36 (2.0 % lower than controls) and remained 2-3 % lower throughout the study, but were identified as statistically significant at around half the time.
These differences in males and females were concurrent with lower bodyweight gains at 1000 mg/kg/day for males and females and males at 500 mg/kg/day. At 12 months, gains were 4.5 and 7.7 % lower for 500 mg/kg/day and 1000 mg/kg/day males, respectively. Bodyweights for females dosed with 1000 mg/kg/day were 2.8 % lower than controls at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Feed consumption for males was found to be increased in the 1000 mg/kg/day dose group at almost every examination and was often statistically significant. This was considered to be an effect of treatment. At 12 months, this was increased by around 3.6 % over control values. Feed consumption for the 5, 50 and 500 mg/kg/day male dose groups was comparable to controls.
Although females fed diet containing the test material tended to have increased feed consumption, this was decreased initially up to day 15. After day 15, feed consumption remained slightly elevated compared to controls up to termination. For doses of 50, 500 and 1000 mg/kg the increase was 2.6, 5.2 and 2.6 % greater than controls, respectively, at termination.
Test material intake was consistent with the target concentrations for all dose levels in the study. Over the course of the entire study, the time weighted average amount of ingested test material (mg/kg/day) was within 2.5 % of the targeted dosages for all dose groups.

FOOD EFFICIENCY
Although feed consumption was highly variable, no clear pattern emerged in relation to administration of the test material.
Feed efficiency decreased over the 13-week time period in both sexes and all dose groups due to slowing of the growth rate while maintaining relatively constant feed consumption. Although slightly increased feed consumption was noted for males given 1000 mg/kg/day and a non-dose related increase for all female dose groups, these minor changes did not result in a consistent pattern of treatment-related altered feed efficiency.

OPHTHALMOSCOPIC EXAMINATION
Ocular haemorrhage, engorged blood vessels, pale fundus, cloudy cornea, periocular soiling, cloudy lens, opaque cornea, opaque lens, phthisis bulbi, missing eye and enlarged or protruding eye were observed. These were not treatment related due to their low incidence and lack of dose-response. Periocular soiling was considered to be a non-specific clinical sign, while eyes with pale fundus, opaque cornea, opaque lens, cloudy cornea or cloudy lens were considered to be age related changes. Phthisis bulbi and missing eye were considered to be secondary to disease or blood collection.

HAEMATOLOGY
No treatment related changes were observed in haematological parameters for treated rats. Females had increased platelets at 5, 500 and 1000 mg/kg/day. This was not dose responsive.
Prothrombin time was equivocally affected for high dose animals and for males only at 500 mg/kg/day. Due to the difference in response between the sexes, the minor differences observed and no association with other clotting disorders, this was not considered to be toxicologically relevant. Males had slightly longer prothrombin times, whereas the affected females had slightly shorter times.

CLINICAL CHEMISTRY
The AST levels of females in the 1000 mg/kg/day group were slightly increased and were attributed to dosing. The AST levels reached statistical significance at 3 and 6 months. This was not considered to be toxicologically significant as this effect had not been demonstrated in previous toxicity studies and the males in this study did not exhibit the same effect despite being the more sensitive of the sexes to effects. This increase also did not accompany any histopathological effects in any organ that could be correlated. There was minimal inter-animal variability in AST levels through 12 month.
Other changes noted in females that were statistically identified were considered to be incidental. Decreased total protein and cholesterol were noted for females at 1000 mg/kg/day at the three month observation. Increased glucose was noted in females at 6 months in the low dose group. Increased cholesterol was observed in the 50 and 500 mg/kg/day groups at 12 months.
There were no electrolyte treatment related changes in either sex at any dose level or time point. Males and females had increased calcium at 500 mg/kg/day for 6 months.

URINALYSIS
At 500 and 1000 mg/kg/day increased urine volume and decreased urine specific gravity, pH, protein and ketones were observed. These effects were treatment related but variable in dose-response relationship and statistical significance. These developed gradually and were less definitive at the end of the study (attributed to geriatric changes). These were unaccompanied by treatment related renal histopathologic changes and were considered to be non-adverse.
The urine changes were considered to be adaptive. The ceca at the two highest doses were enlarged by semi-solid ingesta. These formed normal pellet faeces. This was expected to be as a result in increased colonic water resorption with compensatory renal excretion of the additional water which led to increased urine volume and decreased specific gravity. Decreased urine pH was attributed to renal excretion of the test material. Decreased protein and ketone levels were not considered to be adverse and attributed to the dilution of the normal levels of these normally present in rat urine. Increased urine volume and decreased specific gravity is typically associated with renal disease, however this is usually accompanied by increased proteinuria. Rats dosed with the test material demonstrated less chronic renal disease that that typically associated with male Fischer 344 rats. The renal effects were considered adaptive to altered water balance.

ORGAN WEIGHTS
Both males and females dosed with 500 or 1000 mg/kg/day for 12 months had treatment-related, statistically significant increases in absolute and relative full (including contents) and empty cecal weights. The full cecal weights of males given 500 or 1000 mg/kg/day were approximately 2.3 and 3.8 times greater than the weights of controls, respectively. Females were lesser affected, with the full cecal weights approximately 1.4 and 3.0 times greater than controls for the 500 and 1000 mg/kg/day dose groups, respectively. The empty cecal weights were also found to be increased, but to a lesser degree. Empty cecal weights were increased approximately two-fold for males given 1000 mg/kg/day and approximately 1.7 fold for females.

GROSS PATHOLOGY
The cecum of all males and females dosed 1000 mg/kg/day and 9 males and 6 felaes dose at 500 mg/kg/day was found to be grossly enlarged at study termination. With the exception of optic nerve lesions which were attributed to repetitive retro-orbital bleeding for blood samples, all other observations (including observed masses) were found to be sporadic and not test material related.

HISTOPATHOLOGY: NON-NEOPLASTIC
The only treatment-related histopathologic alteration was a very slight, diffuse hyperplasia of the mucosal epithelium of the cecum of most rats given 1000 mg/kg/day and three males in the 500 mg/kg/day group.
The cecal hyperplasia was associated with enlarged ceca (both in terms of gross observations and organ weight).
All other histopathologic observations were interpreted to be spontaneous alterations, not associated with exposure to the test material with the exception of optic nerve lesions which were again attributed to repetitive retro-orbital bleeding for blood samples.

HISTOPATHOLOGY: NEOPLASTIC
Several tumours were diagnosed for rats but were considered to be unrelated to test material ingestion. Most tumours were benign and were present in only one rat in any dose group. All were considered typical of common spontaneous tumours of Fischer 344 rats.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

50 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were noted in either sex at this level.

2

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg diet

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Slightly decreased bodyweights at 1000 mg/kg/day; Slight diffuse hyperplasia of the cecal mucosal epithelium at 1000 mg/kg/day; Grossly enlarged cecal weights at 500 and 1000 mg/kg/day accompanied by adaptive urinalysis changes.

3

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

other: cecum

Treatment related:

yes

Any other information on results incl. tables

Table 2: Mean bodyweights and selected organ weights

Parameter	Dose Level (mg/kg/day)
	0	5	50	500	1000
Males
Bodyweight (g)	412.5	399.1	405.5	400.8	399.4
Full cecum (g)	3.851	3.373	4.116	8.889*	14.548*
relative (g/100 g)	0.932	0.849	1.015	2.220*	3.647*
Empty cecum (g)	1.328	1.298	1.390	2.203*	2.545*
relative (g/100 g)	0.321	0.326	0.343	0.550*	0.638*
Females
Bodyweight (g)	218.4	228.1	220.0	219.2	207.3
Full cecum (g)	3.790	3.636	3.570	5.284*	11.205*
relative (g/100 g)	1.734	1.600	1.620	2.408*	5.377*
Empty cecum (g)	0.990	0.954	0.965	1.154	1.700*
relative (g/100 g)	0.454	0.422	0.437	0.529	0.819*
* Statistically significant (α = 0.05)

 

Table 3: Selected histopathological changes

Parameter	Dose Level (mg/kg/day)
	0	5	50	500	1000
Males
Very slight diffuse hyperplasia of the cecal mucosa	0	0	0	3	8
Females
Very slight diffuse hyperplasia of the cecal mucosa	0	0	0	0	7

 

Table 4: Mean urine volume (mL)

Time point (months)	Dose Level (mg/kg/day)
	0	5	50	500	1000
Males
3	3.9	3.4	3.6	3.7	5.0
6	3.1	3.3	3.7	5.0*	5.7*
12	4.9	5.1	5.7	9.0*	8.6*
Females
3	2.8	4.2	3.4	4.4*	5.5*
6	3.7	4.3	4.4	5.5	4.8
12	5.7	9.6	6.8	8.1	6.6
* Statistically significant (α = 0.05)

 

Table 5: Mean urine specific gravity

Time point (months)	Dose Level (mg/kg/day)
	0	5	50	500	1000
Males
3	1.081	1.084	1.080	1.083	1.075
6	1.082	1.078	1.075	1.063*	1.064*
12	1.065	1.066	1.061	1.043*	1.049*
Females
3	1.072	1.062	1.062	1.057*	1.052*
6	1.060	1.063	1.057	1.049	1.054
12	1.050	1.041	1.048	1.038	1.044
* Statistically significant (α = 0.05)

Applicant's summary and conclusion

Conclusions:

Administration of the test material was associated with reduced bodyweights, increased cecal weights (empty and full) and hyperplasia of the cecal mucosa in the highest doses tested. The NOEL for toxicity for both sexes was determined to be 50 mg/kg/day as no test material related effects were noted at this level for males and females. The NOAEL for females was determined to be 500 mg/kg/day and the NOAEL for males was 50 mg/kg/day.

Executive summary:

The chronic toxicity of the test material was evaluated in male and female Fischer 344 rats in a study conducted in accordance with the standardised guidelines OECD 453, EU Method B.33, EPA OPPTS 870.4300 and JMAFF Combined Chronic Toxicity/Oncogenicity Study under GLP conditions.

The rats were administered the test material for 1 year in the diet at 0, 5, 50, 500 and 1000 mg/kg/day (10 rats per sex, per dose), which was supplied ad libitum. The animals were observed throughout the study for clinical signs of toxicity, bodyweight and feed consumption, ophthalmological effects, haematological and clinical chemistry changes and at the end of the study underwent gross necropsy with detailed histopathological examinations.

No effects on survival were observed with test material administration, and the animals did not demonstrate clinical signs associated with toxicity. Bodyweights for males and females dosed with 1000 mg/kg/day were found to be lower than controls, as was the bodyweights of male rats dosed with 500 mg/kg/day. In addition to lower bodyweights, 500 and 1000 mg/kg/day males and females had increased absolute and relative full and empty cecal weights, which were observed to be enlarged at gross necropsy. Slight diffuse hyperplasia of the mucosal epithelium of the cecum in male rats was observed at 500 and 1000 mg/kg/day and also in females at 1000 mg/kg/day. The effects noted in the urinalysis results for animals dosed with 500 and 1000 mg/kg/day were considered to be secondary to the affects noted in the cecum.

Administration of the test material was associated with reduced bodyweights, increased cecal weights (empty and full) and hyperplasia of the cecal mucosa in the highest doses tested. The NOEL for toxicity for both sexes was determined to be 50 mg/kg/day as no test material related effects were noted at this level for males and females. The NOAEL for females was determined to be 500 mg/kg/day and the NOAEL for males was 50 mg/kg/day.