2-Pyridinesulfonamide, N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(2,2,2-trifluoroethoxy)-, sodium salt (1:1)

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 1997 to 2 July 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to GLP and in line with standardised guidelines EPA OPP 82-2, OECD 410 and EU method B.9 with no deviations thought to impact on the reliability of the presented results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

sodium 4,6-dimethoxy-N-({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)pyrimidin-2-aminide

EC Number:

688-332-8

Cas Number:

199119-58-9

Molecular formula:

C14H13F3N5O6SNa

IUPAC Name:

sodium 4,6-dimethoxy-N-({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)pyrimidin-2-aminide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: solid (powder)
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

other: Tif:RAIf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 270.8 - 318.1 g (males); 181.5 - 217.7 g (females)
- Housing: individually
- Diet: pelleted, certified standard diet available ad libitum
- Water: tap water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 16 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 23 May 1997 to 2 July 1997

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: carboxymethylcellulose in aqueous polysorbate 80

Details on exposure:

TEST SITE
- Area of exposure: clipped dorsal area
- % coverage: at least 10% of body surface
- Type of wrap if used: test material was applied to gauze patch loosely covered with aluminium wrap, fastened with adhesive but non-irritating tape
- Time intervals for shavings or clippings: day before first application and weekly thereafter

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with lukewarm water
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied: 2.5 mL/kg bodyweight

VEHICLE
- 1% (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80 ( treatment groups 0, 10 and 100 mg/kg test material)
- 0.5% (w/v) carboxymethylcellulose in 0.1% aqueous polysorbate 80 (treatment group 1000 mg/kg test material)

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of test material in the vehicle was carried out at all dose levels on samples collected on experimental days 3, 10, 17 and 24.

The chemical analyses determined the concentrations, homogeneity, and stability of the test material in distilled water with 1% carboxymethylcellulose and 0.1% polysorbate 80. Analysis of homogeneity was performed by analysing samples of each dose group from the top, middle and bottom segments of the mixing container. Evaluation of stability was performed by analysing the samples of each dose after a storage period of seven hours at room temperature. Analysis of concentrations was performed by dissolving the analysis samples (2 g) in 10 mL bi-distilled water with an ultrasonic bath. Solutions were further diluted to 100 mL before a 10 µL aliquot was quantified by HPLC with UV-vis detector at 240 nm.

- Operating conditions
Column dimensions: C18, 5 µm; 125 x 4 mm
Temperature: room temperature
Eluent: methanol/bi-distilled water/buffer solution pH 7.0 (300+650+50 v/v/v)
Flow rate: 1.0 mL/min
Injection volume: 10 µL

Duration of treatment / exposure:

6 hours per day on treatment days

Frequency of treatment:

5 days per week during weeks 1 - 3; 7 days per week during week 4

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 100 and 1000 mg/kg bw
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: all animals were checked daily (morning and afternoon) for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily checks to detect changes in state of health or behaviour, or any reaction to treatment were conducted. Following each application, approximately 17 hours after removal of gauze patches local skin reactions at the application site were assessed according to the Draize Scale.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (on study days -7, 1, 8, 15, 22 and 29)

FOOD CONSUMPTION: Yes
- Time schedule: food consumption was recorded weekly and was calculated for periods of one week. The calculation was based on the weight of the offered diet at the beginning of a weighing period and its difference to the re-weighed amount after several days.

FOOD EFFICIENCY:
Food consumption ratio was calculated using the following: (weekly food consumption (g)/bodyweight (g)) x 1000

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight prior to blood removal)
- How many animals: all surviving animals
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, total white blood cell count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unsustained cells, platelet count, prothrombin time and reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes (overnight prior to blood removal)
- How many animals: all surviving animals
- Parameters checked: urea, creatinine, glucose, albumin, globulin, albumin:globulin ratio, total protein, cholesterol, triglycerides, sodium, potassium, chloride, calcium, total bilirubin, inorganic phosphorous, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were exsanguinated under ether anaesthesia and subjected to detailed necropsy.
- Organ weights: At scheduled termination, the body (exsanguinated), brain, adrenals, heart, kidneys, liver, spleen, ovaries/testes and thymus were removed from all animals and weighed.

HISTOPATHOLOGY: Yes. Skin at application site, skin removal site, mammary area, spleen, mesenteric lymph node, axiliary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary glands, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kidneys, urinary bladder, prostate, seminal vesicle, testes, epididymides, uterus, vagina, ovaries, pituitary gland, adrenal glands, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve, orbital glands, extraorbital lacrimal glands, zymbal glands, muzzle, tongue and any tissue with gross lesions.
- The tissues were preserved in neutral buffered 4% formalin.

MICROSCOPIC EXAMINATIONS: After fixation, a 3-5 µm section of the following organs was taken, embedded, stained and subject to microscopic examination: skin application site, skin removal site, spleen, liver, kidneys, Adrenal glands, thyroid with parathyroid gland and thymus

Any histopathological lesions observed were graded as to degree of severity from grade 1 (minimal/ very few) to grade 5 (massive/ extensive number).

Statistics:

For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods were applied to allow for non normal as well as normal data distribution. Each treated group was compared to the control group by Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean bodyweight gain was significantly decreased (-29%) in the female high dose group.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals died during the study. Furthermore there were no clinical signs were noted and no signs of local irritation occurred.

BODY WEIGHT AND WEIGHT GAIN
The mean bodyweight gain was significantly decreased (-29%) in the female high dose group.

FOOD CONSUMPTION
Food consumption was not influenced by treatment; food consumption by animals in treatment groups was comparable to the controls.

HAEMATOLOGY
Minimally lower mean values for red blood cell count, haemoglobin concentration and haematocrit were recorded for males in the high dose group but without attaining a level of statistical significance. Furthermore, the individual values were within the historical control range. A number of inter-group differences did achieve statistical significance, however, all these differences were accounted for by abnormally high values recorded for male controls which were mainly outside the normal historical control range. In females, small decreases in haemoglobin concentration, haematocrit and clotting activity for animals dosed at 100 mg/kg and a small increase in platelet count in animals dosed at 10 mg/kg attained a level of statistical significance although the differences did not form dose-response relationship and were therefore considered not to be of toxicological relevance.

CLINICAL CHEMISTRY
Differences which attained a level of statistical significance were considered not related to treatment as they did not form a dose-response relationship and/or the magnitude of the change was too small to be of toxicological relevance. In males, these differences included slightly higher plasma chloride values in the 100 and 1000 mg/kg dose groups and slightly lower phosphate in the 10 and 1000 mg/kg dose groups. In females, these differences included minimally lower sodium values for all treated groups with all individual values within the historical control range, and slightly higher alanine aminotransferase activity for the high dose group.

ORGAN WEIGHTS
Statistical significance was reached in absolute and relative thymus weight in males dosed at 100 mg/kg, and relative liver, kidney and spleen weight in males dosed at 10 mg/kg. However, there were no dose-relationships and no other corroborative findings indicative of a treatment-related effect.

GROSS PATHOLOGY
No treatment-related changes were observed at necropsy. Al changes occurred in comparable numbers in all experimental groups and were similar to those occurring spontaneously in the laboratory colony of animals. Thus no experimental relevance was attributed to the findings.

HISTOPATHOLOGY
Ni microscopic lesions were considered treatment-related. All changes observed were considered incidental in nature.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No treatment related effects were noted at this dose level.

2

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Decreased bodyweight gains (-29% compared to controls)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 2: Bodyweight (means) in g/animal

Week	Dose (mg/kg)
	0	10	100	1000
Males
-1	294.0	297.2	290.4	297.2
1	314.8	321.7	311.8	317.8
2	335.4	342.6	329.4	335.6
3	350.1	359.2	338.1	349.4
4	363.7	381.0	359.5	366.2
5	374.5	389.8	365.9	372.4
Females
-1	199.2	195.4	196.9	200.8
1	214.4	212.8	209.2	209.5
2	234.0	230.9	225.4	228.3
3	238.0	240.9	235.2	232.6
4	249.6	248.3	241.8	241.0
5	259.6	255.2	248.5	243.7

 

Table 3: Bodyweight Gain (means) in g/animal

Week	Dose (mg/kg)
	0	10	100	1000
Males
1	20.79	24.50	21.44	20.57
2	41.43	45.36	38.99	38.39
3	56.13	61.96	47.75	52.17
4	69.75	83.80*	69.10	68.99
5	80.50	92.55	75.51	75.19
Females
1	15.21	17.41	12.33	8.706*
2	34.87	35.54	28.54	27.49
3	38.85	45.53	38.38	31.77*
4	50.41	52.93	44.97	40.16
5	60.44	59.84	51.68	42.89*-

 * Wilcoxon if P < 0.05

+- Jonckheere if P < 0.01

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, dermal treatment with the test material was well tolerated. Except for decreased bodyweight gain in females (-29% compared to the control group mean), at the high dose level, no treatment-related effects occurred. There was no irritating effect on the skin.

The NOEL and NOAEL were at 1000 mg/kg bodyweight in males, and at 100 mg/kg bodyweight in females.

Executive summary:

The dermal repeat dose toxicity of the test material was determined in accordance with standardised guidelines EPA OPP 82 -2, OECD 410 and EU Method B.9. Test material was applied under occlusive dressing to the clipped area of skin on the back of rats for a period of 4 weeks on an at least 5 day/week basis. No application was performed on weekend days during treatment weeks 1 -3, but was done on weekends during treatment week 4. The exposure period was 6 hours/day. The test material was suspended in vehicle and was administered at dose levels of 0, 10, 100 and 1000 mg/kg bodyweight per exposure to 5 males and 5 females per dose group. The applied quantities of test material were adjusted weekly to individual animal bodyweight. Control animals were treated with vehicle only. Clinical signs, bodyweight, food consumption and mortality were monitored throughout the study for all animals. Haematology and blood chemistry analyses were performed following the end of treatment. At sacrifice, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Analytical determinations confirmed the test material to be homogeneously distributed in the vehicle and was proven to be stable at the targeted concentrations.

Under the conditions of the study, dermal treatment with the test material was well tolerated. Except for decreased bodyweight gain in females (-29% compared to the control group mean), at the high dose level, no treatment-related effects occurred. There was no irritating effect on the skin.

The NOEL and NOAEL were at 1000 mg/kg bodyweight in males, and at 100 mg/kg bodyweight in females.