1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 Jul 2012 to 14 Sep 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl]benzene-1-sulfonamide

EC Number:

617-298-9

Cas Number:

82097-50-5

Molecular formula:

C14H16ClN5O5S

IUPAC Name:

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl]benzene-1-sulfonamide

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex/Number ordered: 22 males, 22 females. Twenty males and 20 females were assigned to the study. The 2 remaining animals of each sex were assigned as extra animals
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 8-9 weeks old
- Weight at study initiation: 212-250 g for males and 156-194 g for females
- Housing: 2 or 3 per cage by sex in polycarbonate cages (dimensions
61 x 43.5 x 24 cm) with stainless steel grid tops, solid bottoms and an integral food hopper. Sterilised white wood shavings were used as bedding. For environmental enrichment, wooden chewsticks and cardboard play tunnels were supplied and analysis of these items was considered to indicate that there were no substances in sufficient concentration to have any influence on the outcome of the study.
- Diet: Ad libitum with the exception of during the 6 hour period of dermal application where animals had no access to food.
- Water: Water from the public water supply. Ad libitum with the exception of during the 6 hour period of dermal application where animals had no access to water.
- Acclimation period: 14 days. Prior to study commencement, animals were acclimatised to the occlusion procedures over a 5 day period, where the duration of the occlusion was increased on each occasion until the maximum of 6 hours was obtained.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: 10 air changes per hour.
- Photoperiod: 2 h light/dark cycle, light hours being 0700-1900 h

IN-LIFE DATES: From: 10 Jul 2012 To: 14 Sep 2012

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: Milli-Q water

Details on exposure:

TEST SITE
- Area of exposure: lumbar region
- Pre-treatment: The hair on an area of the lumbar region was clipped free of hair as necessary, and test or control substance placed directly onto the skin.
- % coverage: approximately 10 % body surface area. The total body surface area (BSA) of each animal was calculated using the following formula: Total BSA (cm2) = K x W^2/3 Where K = constant for calculating BSA in rats; where W= cubic root of the square of the body weight
- Type of wrap: wetted gauze patches covered in semi-occlusive tape (micropore) and then securely fastened with a strip of occlusive tape (sleek)

REMOVAL OF TEST SUBSTANCE
- The dose site was wiped clean of any excess material, by means of mains supply water and separate gauzes
- Time after start of exposure: 6 hours (± 30 minutes)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hours (± 30 minutes)

Frequency of treatment:

Once daily for at least 28 days and up to the day before termination

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

Three groups of 5 male and 5 female Han Wistar Crl: WI(Han) rats received the test item directly onto the skin in the lumbar region, at dose levels of 10, 100 or 1000 mg/kg bw/day, daily for a period of 6 hours over 28 consecutive days. A concurrent control group was used which received Milli-Q water at a dose volume of 1 mL/kg.
Dose levels for this study, 10, 100 and 1000 mg/kg/day, were selected after evaluation of previous studies carried out by the Sponsor. Previous experience with dermal application of this test substance showed that the material was well tolerated at 1000 mg/kg/day, the limit dose.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
All animals were checked early morning and as late as possible each day for mortality or clinical signs. Once each week, beginning during the week of the pre-trial period, each animal was removed from the cage and received a detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta. Regularly throughout each day, all animals were examined for reaction to treatment. The onset, intensity and duration of any signs were recorded. Particular attention was paid to the animals for the first hour during dosing.

DRAIZE EVALUATION OF DERMAL REACTIONS
Dermal scoring was conducted 0 h (immediately before dosing) and 6 h after each administration of test substance. Skin was assessed for erythema and eschar formation, oedema formation, skin thickening, desquamation and any other reaction to treatment. The scoring system in Table 1 was used for assessing erythema, eschar and oedema formation.

BODY WEIGHT:
Body weights were recorded three times during pre-trial including a Day -1 body weight to allow test substance preparation, and then twice weekly until study completion.

FOOD CONSUMPTION:
The quantity of food consumed by each cage of animals was measured and recorded twice weekly commencing from pre-trial (Day -7) until study completion.

WATER CONSUMPTION:
Water consumption was qualitatively monitored by visual inspection of the water bottles on a weekly basis during the study.

OPHTHALMOSCOPIC EXAMINATION:
The eyes of all animals (including extras) were examined once during the pre-trial period using an indirect ophthalmoscope after the application of mydriatic agent (1% Tropicamide). Anterior, lenticular and fundus areas were evaluated. All control and high dose animals were also examined during Week 4.

HAEMATOLOGY:
Blood samples for haematology, coagulation and clinical chemistry were obtained from all animals, via the orbital sinus under isoflurane anaesthesia on Day 29. Animals were not deprived of food overnight prior to sampling in a random order. Animals were allowed to recover from anaesthesia before terminal necropsy.
-Approximately 0.5 mL of blood was taken into tubes containing EDTA and assayed for the following parameters: Red Blood Cell Count (RBC), Platelets (Plat), Haemoglobin (Hb), Blood smear, Haemoglobin, Distribution Width (HDW), White Blood Cell Count (WBC), Haematocrit (Hct), Neutrophils (Neut), Mean Cell Volume (MCV), Lymphocytes (Lymph), Mean Cell, Haemoglobin Concentration (MCHC),Monocytes (Mono), Mean Cell Haemoglobin (MCH), Eosinophils (Eos), Reticulocytes (Reti %), Basophils (Baso), Reticulocyte Count (absolute) (Ret), Large Unclassified Cells (LUC), Reticulocyte Maturity Index (low, medium and high absorption), Red Cell Distribution Width (RDW).
-Approximately 0.9 mL of blood was collected into tubes containing 3.8% (w/v) trisodium citrate to assess coagulation. The final sample volume was as close as possible to 1.0 mL to give a final concentration of 0.38% (blood to citrate ratio of 9:1). The citrated blood samples were then centrifuged and the plasma separated into plain plastic tubes and analysed for the following parameters: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY:
Approximately 1.0 mL of whole blood was collected and transferred into tubes containing lithium heparin which was then centrifuged and the plasma assayed for the following parameters: Urea (Urea), Chloride (Cl), Urea Nitrogen (BUN), Total Protein (TP), Glucose (Glu), Albumin (Alb), Aspartate Aminotransferase (AST), Globulin (Glob), Alanine Aminotransferase (ALT), Albumin/Globulin ratio (AG-R), Alkaline Phosphatase (ALP), Cholesterol (Chol), Creatine Phosphokinase (CPK), Creatinine (Crea), Lactate Dehydrogenase (LDH), Total Bilirubin (T.Bil), Glutamate Dehydrogenase (GLDH), Calcium (Ca), Gamm-glutamyl Transferase (GGT), Inorganic Phosphate (Phos), Sodium (Na), Triglycerides (Trig), Potassium (K).
The limit of quantification (LOQ) for the following assays was observed and reported as follows with the LOQ used to calculate means and standard deviations for values below the LOQ:
-Gamma-glutamyl Transferase asay; limit of detection: 3 U/L; Non-detectable values reported as: <3 U/L
-Total Bilirubin; limit of detection:1.7 µmol /L; Non-detectable values reported as: <1.7 µmol /L

Sacrifice and pathology:

Following 28 days of treatment, all animals were sacrificed in a random order by exposure to a rising concentration of carbon dioxide and had their terminal body weight recorded followed by severance of major blood vessels.

GROSS PATHOLOGY:
Each animal was subject to a detailed necropsy conducted by a trained technician. A veterinary pathologist was available for consultation for the duration of the necropsy session. The necropsy consisted of a complete external and internal examination including body orifices (ears, nostrils, mouth, anus and vulva) and cranial, thoracic and abdominal organs and tissues. All gross findings were recorded in descriptive terms, including location(s), size (in mm), shape, colour, consistency and number.
The organs marked ‘x’ in the ‘Weighed’ column in Table 2 were removed and weighed from all animals. Paired organs were weighed separately and the sum of the individual organs used for reporting purposes. Representative samples of the tissues listed in Table 2 were taken from all animals and fixed in 10% neutral buffered formalin, unless otherwise stated. Carcasses were discarded after checking the retained tissues against the protocol and a review of the necropsy report.

HISTOPATHOLOGY:
Tissues marked ‘x’ in the ‘Examined’ column in Table 2 were processed to paraffin wax block from all control and high dose animals, sectioned, mounted on glass slides, and stained with haematoxylin and eosin (H&E). Additionally, all gross lesions from low and intermediate dose animals were also processed. Histopathological evaluation of all tissues was then undertaken for all control and high dose animals and for any gross abnormalities (where appropriate) from low and intermediate dose animals.

Other examinations:

BONE MARROW SMEARS
Duplicate bone marrow smears were taken at necropsy and stained using May-Grunwald- Giemsa. No evaluation of the smears was undertaken; smears were retained as a contingency until completion of the study when they were archived.

PEER REVIEW
An internal peer review was undertaken by an appropriately qualified and experienced veterinary pathologist who validated the conclusions of the Study Pathologist by independently assessing the microscope slides. Internal peer review notes are archived with the pathology data

Statistics:

The following statistical approaches were used in this study:
- All analyses were two-tailed for significance levels of 5% and 1%. Males and females were analysed separately.
- All means are presented with standard deviations.
- Body weights, absolute organ weights, haematology, coagulation and clinical chemistry parameters were analysed initially by a one-way analysis of variance (ANOVA).
- Organ weights were also analysed by analysis of covariance (ANCOVA) on final body weight. This statistical analysis provided Adjusted Organ Weight values.
- Summary values of organ to body weight ratios and food consumptions are presented but these were not analysed statistically.
- For all parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control (Group 1) and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant.
- Micropathology incidence data were analysed using Fisher’s Exact Test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no signs indicative of systemic toxicity noted during the observation period. There were local signs recorded at the administration site of several males and females across the dose groups, including controls, where scabbing and/or areas of sparse hair were recorded. Additionally fur staining (2/5) was observed in males that were administered 1000 mg/kg/day of the test item. These local signs were considered minor, transient and were consistent with observations in the control group and as result were not considered to be related to treatment.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

An isolated incidence of very slight erythema (barely perceptible) was recorded in one male (1000 mg/kg/day) on days 5 through 10; mild desquamation (dry skin) was also noted for this animal during this time. This incidence was transient; the condition of the animal’s skin was generally noted to be normal, and this finding is considered to be of doubtful toxicological significance.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related differences in group mean body weights or body weight gain in treated groups in comparison to controls throughout treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related inter-group differences noted in food consumption following administration of the test item at dose levels up to 1000 mg/kg/day.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no ophthalmoscopy findings which were considered to be related to the administration of the test item at dose levels up to 1000 mg/kg/day

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related inter-group differences in haematology or coagulation parameters following administration of the test item at dose levels up to 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related differences in clinical chemistry parameters following administration of the test item at dose levels up to 1000 mg/kg/day.

Statistically significantly higher cholesterol levels were observed in females that received 1000 mg/kg/day when compared with controls. The difference was minor and observed in females only. Due to the isolated nature of the difference it was considered not to be treatment-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related organ weight changes were noted. There were isolated organ weight values that were different from their control. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to differences of sexual maturity and unrelated to administration of the test item

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test item

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test item

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The test item, when administered topically for up to 6 hours daily over a 4 week period, was well tolerated in rats up to 1000 mg/ kg bw/day with no evidence of systemic toxicity. Therefore, under the conditions of this study, the NOAEL was considered to be 1000 mg/kg bw/day.

Executive summary:

This OECD TG 410 study was performed according to GLP, to determine the potential dermal toxicity of the test item when administered topically, for a period of 28 days to rats and to allow hazard classification to be evaluated. Three groups of 5 male and 5 female Han Wistar Crl: WI(Han) rats received the test item directly onto the skin in the lumbar region, at dose levels of 10, 100 or 1000 mg/kg bw/day, daily for a period of 6 hours over 28 consecutive days. A concurrent control group was used which received Milli-Q water at a dose volume of 1 mL/kg. The following parameters and end points were evaluated in this study from all animals: viability, clinical observations, administration-site dermal scoring reactions, body weights, food and water consumption and ophthalmoscopy examinations. Blood samples were collected from all animals at termination for haematology, coagulation and blood chemistry investigations. All animals were terminated after completion of 28 days of treatment and underwent a detailed necropsy examination with selected organs weighed. Tissues from all control and high dose (1000 mg/kg bw/day) animals were subjected to a comprehensive histological examination, with gross lesions (where appropriate) examined from low and intermediate dose animals.

The test item, when administered topically onto the skin for up to 6 hours daily over a 4 week period, was well tolerated in rats up to 1000 mg/ kg bw/day with no evidence of systemic toxicity. There was no impact on body weights, food consumption, blood chemistry or haematological parameters. Additionally, there were no organ weight differences or macroscopic or microscopic local reactions at the administration site. Therefore, the NOAEL was considered to be 1000 mg/kg bw/day.