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

Endpoint summary

Description of key information

ORAL
28-Day
NOAEL = 1000 mg/kg bw/day, rat (male/female), OECD 407, EU Method B.7, EPA OPPTS 870.3050, Stebbins & Day (2000)
NOAEL = 1000 mg/kg bw/day, mouse (male/female), OECD 407, EU Method B.7, EPA OPPTS 870.3050, Yano & Dryzga (2000)
NOAEL = 543 mg/kg bw/day (males); NOEL = 556 mg/kg bw/day (females), dog, Stebbins & Baker (2000)
90-Day
NOAEL = 1000 mg/kg bw/day (females); NOAEL = 500 mg/kg bw/day (males), rat, OECD 408, EU Method B.26, EPA OPPTS 870.3100, JMAFF (2000), Dryzga & Stebbins (2001)
NOEL = 1000 mg/kg bw/day, mouse (male/female), OECD 408, EU Method B.26, EPA OPPTS 870.3100, JMAFF (2000), Stebbins et al. (2001)
NOEL = 232 mg/kg bw/day (females); NOEL = 282 mg/kg bw/day (males), dog, OECD 409, EU Method B.27, EPA OPPTS 870.3150, JMAFF (1985), Stebbins & Baker (2002)
1-Year
NOEL = 50 mg/kg/day (male/female), NOAEL = 500 mg/kg/day (female), NOAEL = 50 mg/kg/day (male), rat, OECD 453, EU Method B.33, EPA OPPTS 870.4300 and JMAFF Combined Chronic Toxicity/Oncogenicity Study, Johnson & Dryzga (2005)
NOEL = 99 mg/kg bw/day (males); NOEL = 93 mg/kg bw/day (females), dog, EPA OPPTS 870.4100, OECD 452, EU Method B (Chronic Toxicity Test) and JMAFF (1-Year Repeated Oral Toxicity Studies), Stebbins & Day (2003)
DERMAL
NOEL(systemic) = 1000 mg/kg bw/day, rat (male/female), OECD 410, EU Method B.9, EPA OPPTS 870.3200, JMAFF (1985), Stebbins et al. (2002)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.4300

Deviations:

no

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

Specific details on test material used for the study:

Purity: 94.5%

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

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.

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.

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.

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

other: cecum

Treatment related:

yes

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)

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Eight repeated dose toxicity studies, conducted via the oral route, are available. Sub-chronic (28 day) studies are available in rats, mice and dogs, sub-chronic (13 week) toxicity studies are available in rats, mice and dogs and 2 chronic (1 year) studies are available in rats and dogs. All studies were conducted under GLP conditions and all bar the 28 day study in dogs were conducted in accordance with standardised guidelines. The overall quality of the database is therefore considered to be high.

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

Study period:

5 March 2002 to 23 December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Testing Guidelines for Toxicology Studies, 1985 (Subchronic Dermal Toxicity Study (Repeated dose dermal toxicity: 21-days study)).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity: 94.5%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: ca. 8 weeks
- Weight at study initiation (group mean): 190.5 - 192.4 g (males); 129.0 - 130.7 g (females)
- Housing: Individually housed in stainless steel cages with wire-mesh floors which were suspended above absorbent cage paper. Cages contained feed containers and pressure activated nipple-type watering systems.
- Diet: ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least 16 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.9 - 22.2 °C
- Humidity: 45.9 - 54.9 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Type of coverage:

semiocclusive

Vehicle:

methylcellulose

Details on exposure:

DERMAL EXPOSURE
Dermal exposure was accomplished by applying the test material to an area that was not less than 10 % of the total body surface. The area on the back of each rat was clipped free of hair at least 24 hours prior to initiation of dosing and on an as needed basis during the dose regime (approximately weekly). An area starting at the scapulae (shoulders) to the wing of the ilium (hipbone) and half way down the flank on each side of the animal was shaved. Animals were acclimated to the semi-occlusion bandages during the three days prior to dosing. The test material or vehicle (0.5 % aqueous MC, dissolved in distilled water) was applied as an aqueous suspension directly to the skin for approximately six hours/day, seven days/week such that a dose volume of 4 mL/kg body weight yielded the appropriate dose. The test material was uniformly applied as a suspension rather than paste due to logistical difficulties with the preparation and accurate application of a paste. Dose amounts were calculated for each animal based on weekly body weights. The vehicle (0.5 % MC) was applied to the backs of control animals at a dose volume of 4 mL/kg. The exposure site was semi-occluded with gauze dressing, non-absorbent cotton, and wrapped in an elastic bandage to hold the test material, gauze dressing and cotton in place. Approximately six hours after application, bandage, gauze and cotton were removed and the exposure site wiped with a water-dampened towel to remove any residual test material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity
Homogeneity analyses of dose suspensions targeted to contain 100 or 1000 mg/kg were determined prior to the start of the study. Samples stored at room temperature were analysed by HPLC with ultraviolet detection and external standard quantification.
The 100 mg/kg/day and 1000 mg/kg/day suspensions were shown to be homogeneously mixed at the study start, with percent relative standard deviations (% RSD) of 0.439 and 1.68 %, respectively.

- Stability
The stability of test material in 0.5% methylcellulose and stored in clear glass containers stored at room temperature was determined prior to the study start by reanalysing concentrations of 100 and 1000 mg/kg 34 days after initial concentration verification. The method used for analysing the test material was a solvent extraction method, followed by analysis using HPLC with ultraviolet detection and external standards.
The test material in the vehicle stored in clear glass vials at room temperature was shown to be stable for at least 34 days. The percent of initial concentration after 34 days was 99.6 %for the 100 mg/kg/day group and 102 % for the 1000 mg/kg/day group.

- Concentration Verification
Analyses of all dose levels, plus control, were conducted prior to the study start. The method used for analysing the test material was a solvent extraction method, followed by analysis using HPLC with ultraviolet detection and external standards.
Results indicated an acceptable agreement between actual and targeted levels with actual values ranging from 105 to 108 % of the targeted concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Animals were dosed six hours/day, seven days per week.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

corresponds to 0 mg/mL

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

corresponds to 25 mg/mL (male) and 26.2 mg/mL (females)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

corresponds to 125 mg/mL (males) and 134.0 mg/mL (females)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

corresponds to 250 mg/mL (males) and 269.0 mg/mL (females)

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high-dose of 1000 mg/kg/day was chosen based on consideration of the findings from existing toxicity studies. This dose also represented the limit test dose level set by several regulatory agencies for dermal toxicity studies. The remaining dose levels were expected to provide dose-response data for any treatment-related effect(s) observed in the high-dose group and to ensure definition of a no-observed-effect level (NOEL).
- Rationale for animal assignment: Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day
- Cage side observations: This examination was performed with the animals in their cages and was designed to: 1) detect significant clinical abnormalities that are clearly visible upon a limited examination and 2) to monitor the general health of the animals. Significant clinical abnormalities that may have been observed included, but were not limited to: changes in activity, repetitive behaviour, vocalisation, incoordination/lameness, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), faecal consistency, and faecal/urinary quantity. At least twice daily all animals were observed for morbidity and mortality, and the availability of feed/water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-exposure and weekly throughout the study.
- Detailed clinical observations: The examination included cage-side, hand-held, and open-field observations that were recorded categorically or using explicitly defined scales.

DERMAL IRRITATION: Yes
- Time schedule for examinations: The dermal test site was subjectively evaluated weekly, using the testing laboratory's modification of the acute dermal irritation scoring system recommended by the Organisation for Economic Co-Operation and Development (see Table 1). In addition, necrosis, scabs and/or scars would have been noted if present. However they would not have been graded.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period and weekly throughout the study. Body weight gains were calculated.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Feed consumption data were collected weekly throughout the study. Feeders were weighed at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) / (number of days in measurement cycle x number of animals in cage)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-exposure and prior to termination using indirect ophthalmoscopy
- Dose groups that were examined: All animals
- Ophthalmoscopic examination: One drop of 0.5% tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during necropsy through a moistened glass slide pressed to the cornea.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Collected from the orbital sinus at scheduled necropsy
- Anaesthetic used for blood collection: Yes (CO₂)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked included: Haematocrit (Hct), Haemoglobin (Hgb) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Platelet (PLAT) count, Differential WBC count, RBC indices (MCH, MCV and MCHC), Prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Collected from the orbital sinus at scheduled necropsy
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked included: Alkaline phosphatase (AP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Albumin (ALB), Cholesterol (CHOL), Creatinine (CREAT), Electrolytes (Na, K, PO₄, Cl and Ca), Glucose (GLU), Total bilirubin (TBILI), Total protein (TP), Urea nitrogen (UN).

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all non-fasted animals during the week prior to necropsy
- Metabolism cages used for collection of urine: Yes (for a 16 hour period)
- Animals fasted: No
- Parameters checked included: Colour, Appearance, Specific gravity, pH, Bilirubin, Glucose, Proteins, Ketones, Blood, Urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

ANIMAL SACRIFICE
Fasted animals submitted alive for necropsy were anaesthetised by the inhalation of CO₂, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanised by decapitation.

GROSS PATHOLOGY: Yes
A complete necropsy was conducted on all animals. The necropsy 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 using a hand-held syringe and blunt needle.
The brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus, cecum (full and empty), and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.

HISTOPATHOLOGY: Yes
Sections from the following tissues, which were preserved in neutral, phosphate-buffered 10 % formalin were processed by standard histologic procedures from control- and high-dose group animals.
Tissues included: adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve - optic, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues/pharynx, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, skin - dermal site, skin - adjacent to dermal test site, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina.
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, lungs, kidneys, dermal test site, skin adjacent to the dermal test site, and relevant gross lesions.

Statistics:

Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, urine specific gravity, clinical chemistry data, coagulation, and appropriate haematologic data were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analyses were performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. Detailed clinical observations incidence scores were statistically analysed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Data collected at different time points were analysed separately. Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices, and differential WBC counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed consumption statistics. Outliers, if identified, were excluded from other analyses only for documented, scientifically sound reasons.
As numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) will be greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results are consistent with other biological and pathological findings and historical control values.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

refer to the field "Details on results" for further information

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY AND CLINICAL SIGNS
There were no deaths during this study.
There were no treatment-related clinical findings during the study. Examinations revealed infrequent occurrences of red periocular soiling, decreased quantity of faeces and focal or multifocal scabs on the skin. Since these observations did not occur in a dose-responsive manner, they were interpreted to not be treatment related.

EVALUATION OF DERMAL SITES
There were no treatment-related gross signs of dermal irritation throughout the entire study for any dose level. One male in the 1000 mg/kg/day group had scabs on the dermal test site on day 28 of study, thought to be associated with the clipping procedure.

BODY WEIGHTS
There were no treatment differences in the body weights or body weight gains of any treated groups when compared to their respective controls.

FEED CONSUMPTION
There were no treatment differences in the feed consumption of any treated groups when compared to their respective controls.

OPHTHALMOLOGY
Examinations performed on all animals pre-exposure and during week 4 revealed no treatment-related findings. Ophthalmic observations (periocular soiling, cloudy cornea, and pale fundus) did not occur in a dose-related manner. Therefore, all ophthalmologic observations were interpreted to be spontaneous alterations.

CLINICAL PATHOLOGY
There were no treatment related changes in any of the haematologic, clinical chemistry, or uninalysis parameters for male and female rats.
Females given 1000 mg/kg/day had a statistically identified decrease in mean cholesterol, relative to controls. However the mean cholesterol value of females given 1000 mg/kg/day (66 mg/dL) was within the historical control range of 58 - 74 mg/dL from other 4-week dermal toxicity studies conducted in the same laboratory, and therefore, was interpreted to be of no toxicological significance.

ANATOMIC PATHOLOGY
> Organ weights: There were no treatment-related changes in any of the organ weight parameters for male and female rats. Males given 100 or 500 mg/kg/day had statistically identified increases in absolute and relative full cecum weights, relative to controls. These alterations in cecum weights were interpreted to not be treatment-related because of the lack of cecal weight increase in males given 1000 mg/kg/day. In addition, there were no statistically identified alterations in empty cecal weights, and there were no histopathologic alterations of the cecum in males and females given 1000 mg/kg/day.
> Gross pathology: There were no treatment-related gross pathologic observations. A few males and females given 0 or 1000 mg/kg/day, and one male given 500 mg/kg/day, had mottling or necrosis of the papillary process of the liver. These gross liver alterations were interpreted to be caused by compressive effects of the elastic bandages used to hold the test material in place. All other gross pathologic observations were considered to be spontaneous alterations, not associated with exposure to the test material.
> Histopathology: The only treatment-related histopathologic observation was slight epidermal hyperplasia at the dermal test site in 2 males given 500 and 3 males given 1000 mg/kg/day. The epidermis of animals with slight hyperplasia was approximately twice as thick as the epidermis of unaffected control animals. There was no microscopic evidence of systemic toxicity at any dose level. A few animals from the control and 1000 mg/kg/day group, and one male given 500 mg/kg/day, had infarction or necrosis with accompanying inflammation of the papillary process of the liver. These liver lesions were interpreted to be caused by compressive effects of the elastic bandages used to hold the test material in place. All other histopathologic observations were considered to be spontaneous alterations, not associated with exposure to test material.

Key result

Dose descriptor:

NOEL

Remarks:

(systemic effects)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic effects at highest dose tested

Critical effects observed:

no

Table 2: Treatment-related Histopathology Findings at the Dermal Test Site

Sex	Males				Females
Dose (mg/kg/day)	0	100	500	1000	0	100	500	1000
Dermal test sites (no. examined)	10	10	10	10	10	10	10	10
Within normal limits	3	2	1	0	7	9	5	6
Hyperplasia, epidermis, very slight	7	8	7	7	3	1	5	4
Hyperplasia, epidermis, slight	0	0	2	3	0	0	0	0

Bold type indicates incidence of effects judged to be treatment-related.

Conclusions:

Under the conditions of this study, the no-observed-effect level (NOEL) for Fischer 344 rats following 28-days of 6 hour/day dermal exposure to the test material was the targeted concentration of 100 mg/kg/day for males and 1000 mg/kg/day for females. The NOEL for systemic effects was 1000 mg/kg/day for both males and females.

Executive summary:

The toxicity of the test material, following repeated dermal exposure, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 410, EU Method B.9, EPA OPPTS 870.3200 and JMAFF (1985).

During the study, ten male and ten female Fischer 344 rats per group were dermally exposed at a semi-occluded skin test site to 0, 100, 500, or 1000 mg test material/kg body weight/day, 6 hours/day, 7 days/week, for at least 28 days. Parameters evaluated were daily cage-side observations, weekly detailed clinical observations, weekly dermal grading, ophthalmologic examinations, body weights, body weight gains, feed consumption, haematology, clinical chemistry, urinalysis, organ weights, and gross and histopathologic examinations. The only treatment-related effect was slight epidermal hyperplasia at the dermal test site of two males given 500 mg/kg/day, and 3 males given 1000 mg/kg/day. The slight epidermal hyperplasia was indicative of minimal irritation in response to dermal application of the test material. Under the conditions of this study, the no-observed-effect level (NOEL) for Fischer 344 rats following 28-days of 6 hour/day dermal exposure to the test material was the targeted concentration of 100 mg/kg/day for males and 1000 mg/kg/day for females. The NOEL for systemic effects was 1000 mg/kg/day for both males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A single study is available. The study was conducted under GLP conditions and in accordance with standardised guidelines. The study was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The overall quality of the dataset is considered to be good.

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

Study period:

5 March 2002 to 23 December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Testing Guidelines for Toxicology Studies, 1985 (Subchronic Dermal Toxicity Study (Repeated dose dermal toxicity: 21-days study)).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity: 94.5%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: ca. 8 weeks
- Weight at study initiation (group mean): 190.5 - 192.4 g (males); 129.0 - 130.7 g (females)
- Housing: Individually housed in stainless steel cages with wire-mesh floors which were suspended above absorbent cage paper. Cages contained feed containers and pressure activated nipple-type watering systems.
- Diet: ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least 16 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.9 - 22.2 °C
- Humidity: 45.9 - 54.9 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Type of coverage:

semiocclusive

Vehicle:

methylcellulose

Details on exposure:

DERMAL EXPOSURE
Dermal exposure was accomplished by applying the test material to an area that was not less than 10 % of the total body surface. The area on the back of each rat was clipped free of hair at least 24 hours prior to initiation of dosing and on an as needed basis during the dose regime (approximately weekly). An area starting at the scapulae (shoulders) to the wing of the ilium (hipbone) and half way down the flank on each side of the animal was shaved. Animals were acclimated to the semi-occlusion bandages during the three days prior to dosing. The test material or vehicle (0.5 % aqueous MC, dissolved in distilled water) was applied as an aqueous suspension directly to the skin for approximately six hours/day, seven days/week such that a dose volume of 4 mL/kg body weight yielded the appropriate dose. The test material was uniformly applied as a suspension rather than paste due to logistical difficulties with the preparation and accurate application of a paste. Dose amounts were calculated for each animal based on weekly body weights. The vehicle (0.5 % MC) was applied to the backs of control animals at a dose volume of 4 mL/kg. The exposure site was semi-occluded with gauze dressing, non-absorbent cotton, and wrapped in an elastic bandage to hold the test material, gauze dressing and cotton in place. Approximately six hours after application, bandage, gauze and cotton were removed and the exposure site wiped with a water-dampened towel to remove any residual test material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity
Homogeneity analyses of dose suspensions targeted to contain 100 or 1000 mg/kg were determined prior to the start of the study. Samples stored at room temperature were analysed by HPLC with ultraviolet detection and external standard quantification.
The 100 mg/kg/day and 1000 mg/kg/day suspensions were shown to be homogeneously mixed at the study start, with percent relative standard deviations (% RSD) of 0.439 and 1.68 %, respectively.

- Stability
The stability of test material in 0.5% methylcellulose and stored in clear glass containers stored at room temperature was determined prior to the study start by reanalysing concentrations of 100 and 1000 mg/kg 34 days after initial concentration verification. The method used for analysing the test material was a solvent extraction method, followed by analysis using HPLC with ultraviolet detection and external standards.
The test material in the vehicle stored in clear glass vials at room temperature was shown to be stable for at least 34 days. The percent of initial concentration after 34 days was 99.6 %for the 100 mg/kg/day group and 102 % for the 1000 mg/kg/day group.

- Concentration Verification
Analyses of all dose levels, plus control, were conducted prior to the study start. The method used for analysing the test material was a solvent extraction method, followed by analysis using HPLC with ultraviolet detection and external standards.
Results indicated an acceptable agreement between actual and targeted levels with actual values ranging from 105 to 108 % of the targeted concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Animals were dosed six hours/day, seven days per week.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

corresponds to 0 mg/mL

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

corresponds to 25 mg/mL (male) and 26.2 mg/mL (females)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

corresponds to 125 mg/mL (males) and 134.0 mg/mL (females)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

corresponds to 250 mg/mL (males) and 269.0 mg/mL (females)

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high-dose of 1000 mg/kg/day was chosen based on consideration of the findings from existing toxicity studies. This dose also represented the limit test dose level set by several regulatory agencies for dermal toxicity studies. The remaining dose levels were expected to provide dose-response data for any treatment-related effect(s) observed in the high-dose group and to ensure definition of a no-observed-effect level (NOEL).
- Rationale for animal assignment: Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day
- Cage side observations: This examination was performed with the animals in their cages and was designed to: 1) detect significant clinical abnormalities that are clearly visible upon a limited examination and 2) to monitor the general health of the animals. Significant clinical abnormalities that may have been observed included, but were not limited to: changes in activity, repetitive behaviour, vocalisation, incoordination/lameness, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), faecal consistency, and faecal/urinary quantity. At least twice daily all animals were observed for morbidity and mortality, and the availability of feed/water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-exposure and weekly throughout the study.
- Detailed clinical observations: The examination included cage-side, hand-held, and open-field observations that were recorded categorically or using explicitly defined scales.

DERMAL IRRITATION: Yes
- Time schedule for examinations: The dermal test site was subjectively evaluated weekly, using the testing laboratory's modification of the acute dermal irritation scoring system recommended by the Organisation for Economic Co-Operation and Development (see Table 1). In addition, necrosis, scabs and/or scars would have been noted if present. However they would not have been graded.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period and weekly throughout the study. Body weight gains were calculated.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Feed consumption data were collected weekly throughout the study. Feeders were weighed at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) / (number of days in measurement cycle x number of animals in cage)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-exposure and prior to termination using indirect ophthalmoscopy
- Dose groups that were examined: All animals
- Ophthalmoscopic examination: One drop of 0.5% tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during necropsy through a moistened glass slide pressed to the cornea.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Collected from the orbital sinus at scheduled necropsy
- Anaesthetic used for blood collection: Yes (CO₂)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked included: Haematocrit (Hct), Haemoglobin (Hgb) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Platelet (PLAT) count, Differential WBC count, RBC indices (MCH, MCV and MCHC), Prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Collected from the orbital sinus at scheduled necropsy
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked included: Alkaline phosphatase (AP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Albumin (ALB), Cholesterol (CHOL), Creatinine (CREAT), Electrolytes (Na, K, PO₄, Cl and Ca), Glucose (GLU), Total bilirubin (TBILI), Total protein (TP), Urea nitrogen (UN).

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all non-fasted animals during the week prior to necropsy
- Metabolism cages used for collection of urine: Yes (for a 16 hour period)
- Animals fasted: No
- Parameters checked included: Colour, Appearance, Specific gravity, pH, Bilirubin, Glucose, Proteins, Ketones, Blood, Urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

ANIMAL SACRIFICE
Fasted animals submitted alive for necropsy were anaesthetised by the inhalation of CO₂, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanised by decapitation.

GROSS PATHOLOGY: Yes
A complete necropsy was conducted on all animals. The necropsy 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 using a hand-held syringe and blunt needle.
The brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus, cecum (full and empty), and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.

HISTOPATHOLOGY: Yes
Sections from the following tissues, which were preserved in neutral, phosphate-buffered 10 % formalin were processed by standard histologic procedures from control- and high-dose group animals.
Tissues included: adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve - optic, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues/pharynx, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, skin - dermal site, skin - adjacent to dermal test site, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina.
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, lungs, kidneys, dermal test site, skin adjacent to the dermal test site, and relevant gross lesions.

Statistics:

Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, urine specific gravity, clinical chemistry data, coagulation, and appropriate haematologic data were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analyses were performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. Detailed clinical observations incidence scores were statistically analysed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Data collected at different time points were analysed separately. Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices, and differential WBC counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed consumption statistics. Outliers, if identified, were excluded from other analyses only for documented, scientifically sound reasons.
As numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) will be greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results are consistent with other biological and pathological findings and historical control values.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

refer to the field "Details on results" for further information

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY AND CLINICAL SIGNS
There were no deaths during this study.
There were no treatment-related clinical findings during the study. Examinations revealed infrequent occurrences of red periocular soiling, decreased quantity of faeces and focal or multifocal scabs on the skin. Since these observations did not occur in a dose-responsive manner, they were interpreted to not be treatment related.

EVALUATION OF DERMAL SITES
There were no treatment-related gross signs of dermal irritation throughout the entire study for any dose level. One male in the 1000 mg/kg/day group had scabs on the dermal test site on day 28 of study, thought to be associated with the clipping procedure.

BODY WEIGHTS
There were no treatment differences in the body weights or body weight gains of any treated groups when compared to their respective controls.

FEED CONSUMPTION
There were no treatment differences in the feed consumption of any treated groups when compared to their respective controls.

OPHTHALMOLOGY
Examinations performed on all animals pre-exposure and during week 4 revealed no treatment-related findings. Ophthalmic observations (periocular soiling, cloudy cornea, and pale fundus) did not occur in a dose-related manner. Therefore, all ophthalmologic observations were interpreted to be spontaneous alterations.

CLINICAL PATHOLOGY
There were no treatment related changes in any of the haematologic, clinical chemistry, or uninalysis parameters for male and female rats.
Females given 1000 mg/kg/day had a statistically identified decrease in mean cholesterol, relative to controls. However the mean cholesterol value of females given 1000 mg/kg/day (66 mg/dL) was within the historical control range of 58 - 74 mg/dL from other 4-week dermal toxicity studies conducted in the same laboratory, and therefore, was interpreted to be of no toxicological significance.

ANATOMIC PATHOLOGY
> Organ weights: There were no treatment-related changes in any of the organ weight parameters for male and female rats. Males given 100 or 500 mg/kg/day had statistically identified increases in absolute and relative full cecum weights, relative to controls. These alterations in cecum weights were interpreted to not be treatment-related because of the lack of cecal weight increase in males given 1000 mg/kg/day. In addition, there were no statistically identified alterations in empty cecal weights, and there were no histopathologic alterations of the cecum in males and females given 1000 mg/kg/day.
> Gross pathology: There were no treatment-related gross pathologic observations. A few males and females given 0 or 1000 mg/kg/day, and one male given 500 mg/kg/day, had mottling or necrosis of the papillary process of the liver. These gross liver alterations were interpreted to be caused by compressive effects of the elastic bandages used to hold the test material in place. All other gross pathologic observations were considered to be spontaneous alterations, not associated with exposure to the test material.
> Histopathology: The only treatment-related histopathologic observation was slight epidermal hyperplasia at the dermal test site in 2 males given 500 and 3 males given 1000 mg/kg/day. The epidermis of animals with slight hyperplasia was approximately twice as thick as the epidermis of unaffected control animals. There was no microscopic evidence of systemic toxicity at any dose level. A few animals from the control and 1000 mg/kg/day group, and one male given 500 mg/kg/day, had infarction or necrosis with accompanying inflammation of the papillary process of the liver. These liver lesions were interpreted to be caused by compressive effects of the elastic bandages used to hold the test material in place. All other histopathologic observations were considered to be spontaneous alterations, not associated with exposure to test material.

Key result

Dose descriptor:

NOEL

Remarks:

(systemic effects)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic effects at highest dose tested

Critical effects observed:

no

Table 2: Treatment-related Histopathology Findings at the Dermal Test Site

Sex	Males				Females
Dose (mg/kg/day)	0	100	500	1000	0	100	500	1000
Dermal test sites (no. examined)	10	10	10	10	10	10	10	10
Within normal limits	3	2	1	0	7	9	5	6
Hyperplasia, epidermis, very slight	7	8	7	7	3	1	5	4
Hyperplasia, epidermis, slight	0	0	2	3	0	0	0	0

Bold type indicates incidence of effects judged to be treatment-related.

Conclusions:

Under the conditions of this study, the no-observed-effect level (NOEL) for Fischer 344 rats following 28-days of 6 hour/day dermal exposure to the test material was the targeted concentration of 100 mg/kg/day for males and 1000 mg/kg/day for females. The NOEL for systemic effects was 1000 mg/kg/day for both males and females.

Executive summary:

The toxicity of the test material, following repeated dermal exposure, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 410, EU Method B.9, EPA OPPTS 870.3200 and JMAFF (1985).

During the study, ten male and ten female Fischer 344 rats per group were dermally exposed at a semi-occluded skin test site to 0, 100, 500, or 1000 mg test material/kg body weight/day, 6 hours/day, 7 days/week, for at least 28 days. Parameters evaluated were daily cage-side observations, weekly detailed clinical observations, weekly dermal grading, ophthalmologic examinations, body weights, body weight gains, feed consumption, haematology, clinical chemistry, urinalysis, organ weights, and gross and histopathologic examinations. The only treatment-related effect was slight epidermal hyperplasia at the dermal test site of two males given 500 mg/kg/day, and 3 males given 1000 mg/kg/day. The slight epidermal hyperplasia was indicative of minimal irritation in response to dermal application of the test material. Under the conditions of this study, the no-observed-effect level (NOEL) for Fischer 344 rats following 28-days of 6 hour/day dermal exposure to the test material was the targeted concentration of 100 mg/kg/day for males and 1000 mg/kg/day for females. The NOEL for systemic effects was 1000 mg/kg/day for both males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.76 mg/cm²

Study duration:

subacute

Species:

rat

Quality of whole database:

A single study is available. The study was conducted under GLP conditions and in accordance with standardised guidelines. The study was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). The overall quality of the dataset is considered to be good.

Additional information

ORAL

A number of repeated dose toxicity studies are available in which animals were dosed via the oral route. In all cases the animals received test material in the diet. Each available study is summarised below. With the exception of the 28-day study conducted in the dog (which was awarded a reliability score of 2) all studies were awarded a reliability score of 1 in accordance with the criteria of Klimisch et al. (1997).

In the key study, Johnson & Dryzga (2005), 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.

Sub-Acute (28-Day Exposure)

Rat

The toxicity of the test material was assessed following repeated exposure via the dietary route. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 407, EU Method B.7, and EPA OPPTS 870.3050.

During the study five Fischer 344 rats/sex/dose group were given test diets formulated to supply 0, 10, 100, 500 or 1000 mg test material/kg bw/day, for 4 weeks. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, test material intake, haematology (including prothrombin time), clinical chemistry, urinalysis, selected organ weights, gross and histopathologic examinations.

There were no treatment-related effects in body weights, feed consumption, ophthalmologic and clinical observations, organ weights or clinical pathology parameters. The only treatment-related gross observation was an increased size of the cecum, noted in 3 males and 2 females given 500 mg/kg/day, and 5 males and 5 females given 1000 mg/kg/day. There were no histopathologic changes associated with the cecal alteration. Therefore, the increased size of the cecum was interpreted to be a non-adverse effect, reflective of physiological changes in the digestive tract following ingestion of the test material. There were no treatment-related histopathologic observations.

Based on the increased size of the cecum noted in rats given 500 mg/kg/day or 1000 mg/kg/day, the no-observed-effect level (NOEL) for Fischer 344 rats of either sex following 4-weeks of dietary exposure to test material was 100 mg/kg/day. The no-observed-adverse- effect level (NOAEL) was 1000 mg/kg/day for both sexes.

A wealth of good quality data was available as supporting information, and is summarised below.

Mouse

The toxicity of the test material was assessed following repeated exposure via the dietary route. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 407, EU Method B.7, and EPA OPPTS 870.3050.

During the study, five male and five female CD-1 mice per group were given test diets formulated to supply 0, 10, 100, 500 or 1000 mg of test material per kilogram body weight per day (mg/kg/day) for 4 weeks. Standard toxicologic parameters were evaluated.

There were no treatment-related effects in clinical observations, body weights, feed consumption, test material intake, haematology, clinical chemistry, organ weight or gross pathologic parameters. Treatment related histopathologic effects were limited to two of five male mice given 1000 mg/kg/day and consisted of an increase in hepatocyte size with altered cytoplasmic staining of the cytoplasm and a decrease in glycogen.

Therefore, under the conditions of the study, the no observed-adverse-effect level (NOAEL) was 1000 mg/kg/day for male and female CD-1 mice, and the no-observed-effect level (NOEL) was determined to be 500 mg/kg/day for males and 1000 mg/kg/day for females.

Dog

The toxicity of the test material was assessed in a study which was conducted under GLP conditions, following repeated exposure to dogs dosed via the dietary route for a period of 4 weeks.

During the study male and female Beagle dogs (two/sex/dose level) were fed diets formulated to contain 0, 0.15, 0.45, or 1.5 % test material. These concentrations corresponded to dose levels of approximately 0, 62, 193, or 543 mg/kg/day in males, and 0, 62, 177, or 556 mg/kg/day in females. Parameters evaluated were clinical appearance, ophthalmologic examinations, body weight, feed consumption, clinical chemistry, haematology, urinalysis, selected organ weights, gross and histopathologic examinations.

Males dogs given 1.5 % test material had slightly lower feed consumption, relative to controls, during weeks 2-4. The feed consumption of males given 0.15 % or 0.45 %, and all female treated groups, was comparable to controls. There were no treatment-related effects in any of the remaining study parameters listed above.

Under the conditions of this study, the no-observed-effect level (NOEL) for females was 1.5 % (556 mg/kg/day) and 0.45 % (193 mg/kg/day) for males, based on slightly lower feed consumption of males at the 1.5 % dose level. The no-observed-adverse-effect level (NOAEL) for males was 1.5 % (543 mg/kg/day).

Sub-Chronic (90-Day (13 Week) Exposure)

Rat

The toxicity of the test material following repeated dietary exposure, to rats, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 408, EU Method B.26, EPA OPPTS 870.3100 and JMAFF (2000).

During the study, ten male and ten female Fischer 344 rats per group were given test diets formulated to supply 0, 10, 100, 500, or 1000 milligrams of test material per kilogram body weight per day (mg/kg/day) for 13 weeks. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, haematology (including prothrombin time), clinical chemistry, urinalysis, selected organ weights, gross and histopathologic examinations. An additional ten male and ten female rats in the control and high-dose groups were held untreated for four weeks following the dosing period to assess recovery from treatment-related effects.

Following the 13-week exposure period, there were no treatment-related effects in body weights, feed consumption, ophthalmologic and clinical observations, haematologic and clinical chemistry parameters. Males given 1000 mg/kg/day had treatment-related hyperplasia of the mucosal epithelium of the cecum and ileum. The hyperplasia of the cecal epithelium corresponded to a statistically-identified increase in mean cecal weight for males given 1000 mg/kg/day. Males given 500 mg/kg/day and females given 500 or 1000 mg/kg/day also had treatment-related increases in cecal weights, however, there were no corresponding microscopic alterations of the cecum or ileum in these animals. The only other treatment-related alterations were decreases in urine pH for males and females given 500 or 1000 mg/kg/day, and decreases in urine protein and ketones for males and females given 1000 mg/kg/day.

Following the 4-week recovery period there was complete resolution of the hyperplasia of the mucosal epithelium of the cecum and ileum, and partial recovery of the increased cecal weights of males and females from the 1000 mg/kg/day group. In addition, there was complete recovery of the urine alterations in males and females from the 1000 mg/kg/day group.

Based on changes in cecal weight and urine pH, the no-observed-effect level (NOEL) for male and female Fischer 344 rats was the targeted dietary dose of 100 mg/kg/day. At 500 mg/kg/day for males, and 1000 mg/kg/day for females, increases in cecal weights and decreases in urine pH were unaccompanied by microscopic alterations. Therefore, the no-observed-adverse-effect level (NOAEL) for males was 500 mg/kg/day, and the NOAEL for females was 1000 mg/kg/day.

Mouse

The toxicity of the test material was investigated following repeated dietary exposure, to mice, over a period of 13 weeks. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 408, EU Method B.26, EPA OPPTS 870.3100 and JMAFF (2000).

During the study ten male and ten female CD-1 mice per group were given test diets formulated to supply 0, 10, 100, 500 or 1000 milligrams of test material per kilogram body weight per day (mg/kg/day) for 13 weeks. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, haematology, clinical chemistry, selected organ weights, and gross and histopathologic examinations.

There were no treatment-related effects in any of the parameters. Based on the multiple parameters evaluated in this study, the no-observed-effect level (NOEL) for CD-1 mice of either sex was the limit test concentration of 1000 mg/kg/day.

Dog

The toxicity of the test material following 13 weeks of repeated dietary exposure was investigated under GLP conditions and in accordance with the standardised guidelines OECD 409, EU Method B.27, EPA OPPTS 870.3150 and JMAFF (1985).

During the study male and female Beagle dogs (four/sex/dose level) were fed diets formulated to contain 0, 0.15, 0.75, or 3.0 % test material. These concentrations corresponded to dose levels of approximately 54.5, 282, and 1070 mg/kg/day in males, and 52.7, 232, and 929 mg/kg/day in females. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, haematology (including prothrombin time), clinical chemistry, urinalysis, selected organ weights, gross and histopathologic examinations.

There were no treatment-related effects on body weights, feed consumption, ophthalmologic and clinical observations, organ weights or clinical pathology parameters. A treatment-related microscopic effect was noted in the stomachs of all males and females given 3.0 % test material. The effect was characterised by slight, diffuse hyperplasia and hypertrophy of mucous cells, and slight, diffuse hyperplasia of chief cells in the gastric mucosa. There were no treatment-related effects in males or females given 0.15 or 0.75 %. The no-observed-effect level (NOEL) in male and female Beagle dogs following 13- weeks of dietary exposure was therefore 0.75 % test material which is equivalent to 232 mg/kg/day in female dogs and 282 mg/kg/day in male dogs.

Chronic (1 Year Exposure)

Dog

The toxicity of the test material was investigated following repeated dietary exposure, for a period of 1 year, to Beagle dogs. The study was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.4100, OECD 452, EU Method B (Chronic Toxicity Test) and JMAFF (1-Year Repeated Oral Toxicity Studies).

During the study male and female dogs (four/sex/dose) were fed diets formulated to provide 0, 0.03, 0.3, or 3.0 % test material. These concentrations corresponded to approximately 0, 9.9, 99.2, and 967 mg/kg/day in males, and 0, 9.2, 93.2, and 1038 mg/kg/day in females. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, haematology, clinical chemistry, urinalysis, selected organ weights, and gross and histopathologic examinations.

There were no treatment-related effects on feed consumption, ophthalmologic and clinical observations, or clinical pathology parameters. High-dose (3.0 % test material) females had treatment-related lower final body weights and body weight gains. High-dose animals had treatment-related, statistically identified higher relative liver weights averaged across both sexes, relative to controls. In addition, there was a treatment related non-statistically identified increase in the mean absolute liver weight of high dose males, relative to controls. The liver weight alterations corresponded to very slight hypertrophy of centrilobular to midzonal hepatocytes in two males and two females from the high-dose group. Treatment-related microscopic effects were noted in the stomachs of all high-dose males and females. The stomach effects consisted of slight, diffuse mucosal hyperplasia and hypertrophy, very slight or slight chronic mucosal inflammation, and slight lymphoid hyperplasia of the gastric mucosa. There were no treatment-related effects in males or females given 0.03 or 0.3% test material.

Therefore, under the conditions of the study, the no-observed-effect level (NOEL) in male and female Beagle dogs following one year of dietary exposure was 0.3% test material, which corresponded to 99 and 93 mg/kg/day, respectively.

DERMAL

The toxicity of the test material, following repeated dermal exposure, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 410, EU Method B.9, EPA OPPTS 870.3200 and JMAFF (1985).

During the study, ten male and ten female Fischer 344 rats per group were dermally exposed at a semi-occluded skin test site to 0, 100, 500, or 1000 mg test material/kg body weight/day, 6 hours/day, 7 days/week, for at least 28 days. Parameters evaluated were daily cage-side observations, weekly detailed clinical observations, weekly dermal grading, ophthalmologic examinations, body weights, body weight gains, feed consumption, haematology, clinical chemistry, urinalysis, organ weights, and gross and histopathologic examinations. The only treatment-related effect was slight epidermal hyperplasia at the dermal test site of two males given 500 mg/kg/day, and 3 males given 1000 mg/kg/day. The slight epidermal hyperplasia was indicative of minimal irritation in response to dermal application of the test material. Under the conditions of this study, the no-observed-effect level (NOEL) for Fischer 344 rats following 28-days of 6 hour/day dermal exposure to the test material was the targeted concentration of 100 mg/kg/day for males and 1000 mg/kg/day for females. The NOEL for systemic effects was 1000 mg/kg/day for both males and females.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The key study was selected as it was a chronic study, performed on the preferred animal model which resulted in the lowest NOAEL for assessment.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Only one study is available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Only one study is available.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: cecum

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to repeated dose toxicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to repeated dose toxicity.