Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 July 2015 to 14 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP and OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Devations:Deviation No. 1The weight in females (150.04-170.06 g) and some males (241.60-244.75 g) on day 1 of treatment were not within the range specified in the study plan (males: 198-238 g and females: 125 150 g). The weight of females at arrival was higher than expected and no study plan amendment was issued.Deviation No. 2The push-pull strain gauge to determine the grip strength used (BIOSEB GT3 no. BT0171) is not the same as that stated in the Study plan (Mecmesin, AFG 25N).Deviation No. 3On 23 July 2015 (day 2 of acclimatization), room temperature and humidity were not monitored for 7 minutes due to an incidence in the SCADA monitoring system.Values outside the established ranges for humidity (30-70%) occurred occasionally (around 95%).Deviation No. 4The blood sample obtained from some animals (1M, 4M, 12M, 43F, 52F) for coagulation parameters was coagulated and although the extraction was repeated, no results for PT or APTT are reported for animals 12M, 43F or 52F. The samples were coagulated again and not analyzed.The sample from females 67 and 68 was also coagulated and not analyzed; no repetitions were done to avoid obtaining more blood and therefore no results are reported for PT or APTT.Deviation No. 5A sample from the tongue was fixed in formalin solution and a slide prepared although this was not indicated in the study plan; therefore, it will not be examined histopathologically. Deviation No. 6Given the results of the dose formulation prepared at the low dose (19 October) and to be administered in week 13, a new analysis was done. New samples were taken and frozen on 27 October from a formulation prepared on 23 October. Aliquot 1 was analyzed, but it was not possible to analyze aliquot 2 (bottom) as, according to the Principal Investigator, the tube did not contain any sample although according to the raw data the sample had been taken. Given the nature and magnitude of these deviations, it can be stated that they did not alter the conclusions of the Study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

EC Number:

223-989-2

EC Name:

Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

Cas Number:

4156-21-2

Molecular formula:

C9H6Cl2N4O3S.Na

IUPAC Name:

sodium 4-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Details on test material:

Identification: Granofin Easy F-90Chemical Name: Sodium p-[(4,6-dichloro- 1,3,5-triazin-2yl)amino]benzenesulphonateCAS no.: 4156-21-2

Test animals

Species:

rat

Strain:

other: Han Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test SystemAnimals:Han Wistar ratsRationale:Recognized by the international guidelines as a recommended test systemSupplier:Charles River Laboratories (Germany)Source:Charles River Laboratories, Sandhofer Weg 7, 97633 Sulzfeld,GermanyNumber of Animals:90 (45 males and 45 females) were received, of which 10 males and 10 females were allocated to each treatment group ( see also Section 3.3).Sex: Males and females, the females were nulliparous and nonpregnantTotal Number of Animals per Group:Groups 1 to 4: 10 males and 10 females eachAge (at Treatment Start):7-9 weeksBody-weight Range (at Treatment Start):Males: 205.33 – 244.75 g Females: 150.04 – 170.06 gAt the commencement of the treatment, the weight differences of the animals used was not more than ±20% the mean weight of each sex.Identification:Cage card and tail mark (later ear tattoo)Randomization:Method based on the similarity of mean body weights among groupsAcclimatization:8 days Veterinary Inspection:At delivery (23 July 2015) and prior to commencement of dosing (28 July 2015), the animals were examined by a veterin ary surgeon. The animals used in the study had no visible signs of illness.AllocationThe animals were allocated to the four treatment groups and assigned numbers 1 to 80. The spare animals were allocated to group 5 and assigned numbers 81 to 90. Shortly before the start of treatment, the allocation was adjusted where considered appropriate using spare animals. The rejected animals took no further part in the study after the start of treatment and were removed from the study. Only data from animals assigned to the study are included in the report. The permanent group identification and animal numbers assigned for treatment (groups 1 to 4) are indicated in the following table.HusbandryDiet:Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Harlan Laboratories Models, S.L.). The batches and expiry dates are indicated below: 010615MA, 3 October 2015 051915MB, 13 February 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

prepared in-house with Milli-Q water purification system (MILLI Q Gradient A10 Century, Merck)

Details on oral exposure:

Dose Formulation PreparationThe dose formulations were prepared up to six days in advance. Dose levels were calculated based on the active ingredient. Granofin Easy F-90 was weighed in a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). A homogeneous suspension was prepared using a magnetic stirrer. Having obtained a homogeneous mixture, the suspension was transferred to a volumetric flask and filled to the mark using the remaining vehicle. The formulation was transferred to a glass beaker and when there was enough vehicle left for the final formulation volume, it was used to clean the volumetric flask and was then added to the glass beaker.The total volume to be administered on several consecutive days was prepared (stock solution) in advance and the necessary amount aliquoted for each administration day.Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.Dose formulations were stored in the refrigerator (5 ± 3 °C) in suitable glass containers appropriate for the administration. They were allowed to reach room temperature just before dosing.Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study D51272 14-Day Oral Toxicity (Gavage) Study in the Wistar Rat, dose formulations are stable for at least seven days if stored in the refrigerator.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations prepared and administered in weeks 1 and 13 were analyzed for homogeneityand concentrationThe formulations were analyzed using the HPLC with UV detection method provided by Harlan Laboratories Ltd and adapted at Envigo CRS Limited (Study Number TMR0143).

Duration of treatment / exposure:

90 or 91 days. The animals were sacrificed in two days i.e. the animals sacrificed on the second day remained 91 days on treatment.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:5 mg/Kg bwBasis:actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:25 mg/Kg bwBasis:actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:100 mg/Kg bwBasis:actual ingested

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Viability / Mortality: Cage-side clinical observations were performed twice dailyClinical Signs: Detailed clinical signs were recorded on the first day of treatment and daily from the third day of administration. These observations we re done at the time of administration: Animals were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secreti ons and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also che ckedFunctional Observational Battery (Sensory Activity, Grip strength assessment, Locomotor activity assessment), on the day of sacrifice, Days 85 and 86Food Consumption: Before treatment start (days 3-9 of the acclimatization period), and once a week during treatment periodBody Weight: Once before treatment start, once weekly during treatment and before sacrificeOphthalmoscopy (Cornea, lens, conjunctiva, sclera, iris and fundus): on Day 7 for all animals and on Day 89, just for group 4, because no treatment-r elated alterations were recorded in group 4. Clinical Laboratory Investigations (Hematology, Coagulation, Blood Biochemistry): on the day of sacrifice ( Days 91 and 92)Pathology (Sacrifice and Macroscopic Examinations, Organ Weights, Histopathology)

Sacrifice and pathology:

At the end of the treatment period, all surviving animals were deprived of food overnight and were deeply anesthetized with sodium pentobarbital administered intraperitoneally and then exsanguinated by excision of the axillary vessels and aorta.

Statistics:

The following statistical methods were used by the ToxControl system Version 8.0 to analyze body weight, food consumption, grip strength, locomotor activity, clinical laboratory data and organ weights and ratios as well as macroscopic findings:1.The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied when the variables were assumed to follow a normal dist ribution for the comparison of the treated groups and the control groups for each sex (Dunnett, 1955).2.The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution (Miller, 1981).3.Fisher's exact-test was applied to the macroscopic findings (Fisher, 1950).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The animals survived the scheduled treatment period. No clinical signs were recorded attributed to treatmentSlightly increased salivation was observed just after administration for a maximum of 5 minutes often during treatment in most group-4 animals and just once in five group-3 animals. Occasionally some of the animals from group 4 started to show the increase just before or during administration. Given the moment in which it was observed and the duration it is considered due to the handling rather than to an effect of the test item. Some animals from groups 3 (26M, 28M, 29M) and 4 (36M, 38M, 39M, 40M, 77F, 78F, 79F) showed a wound or scab and hypotrichosis and/or alopecia on an small area of the face. Female 66 (group 3) showed hypotrichosis in the abdominal area. In most of the cases, these alterations were observed from weeks 9-10 and were not observed at the end of treatment. Given that this sign is often observed in rats and given the incidence, they are not attributed to an effect of the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

The animals survived the scheduled treatment period. No clinical signs were recorded attributed to treatmentSlightly increased salivation was observed just after administration for a maximum of 5 minutes often during treatment in most group-4 animals and just once in five group-3 animals. Occasionally some of the animals from group 4 started to show the increase just before or during administration. Given the moment in which it was observed and the duration it is considered due to the handling rather than to an effect of the test item. Some animals from groups 3 (26M, 28M, 29M) and 4 (36M, 38M, 39M, 40M, 77F, 78F, 79F) showed a wound or scab and hypotrichosis and/or alopecia on an small area of the face. Female 66 (group 3) showed hypotrichosis in the abdominal area. In most of the cases, these alterations were observed from weeks 9-10 and were not observed at the end of treatment. Given that this sign is often observed in rats and given the incidence, they are not attributed to an effect of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Although the statistical significant differences in females from group 4 were higher compared to the control group during most of the treatment, no relevant variations are observed in body weigh gain and increases were similar between groups at the end

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No relevant differences were recorded between groups.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

No alterations attributed to treatment were recorded. One female from group 4 showed redness of the retina in the right eye but it is considered an occasional finding

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No alterations attributed to treatment were recorded. PT and APPTT values were higher in 65F (group 3) than in the rest of the animals but the sample was partially coagulated.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No alterations attributed to the treatment were recorded

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No alterations attributed to the treatment were recorded

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

No alterations attributed to the treatment were recorded

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No alterations attributed to the treatment were recorded

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No alterations attributed to the treatment were recorded

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No alterations attributed to the treatment were recorded

Effect levels

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, oral administration of Granofin Easy F-90 to Wistar rats during 90 consecutive days did not induce alterations or toxicological effects. Therefore, the dose of 100 mg/kg/day is considered the NOEL (No Observed Effects Level).

Executive summary:

Granofin Easy F-90 wasadministered orally (by gavage) to Wistar rats once daily for 90 days. Four treatment groups were established, each containing 10 males and 10 females.

 

Animals from group 1 were administered with the vehicle (purified water) following the same dosing regimen as the groups treated with the test item and animals from groups 2, 3 and 4 with the test item at 5, 25 and 100 mg/kg, respectively.

 

Dose formulations prepared and administered in weeks 1 and 13 were analyzed for homogeneity and concentration.

 

After the treatment period, all animals were necropsied and various organs were weighed. Macroscopic alterations were recorded. A full set of tissues and organs was collected but only samples taken from groups 1 and 4 were processed and examined histopathologically. Samples form groups 2 and 3 were not processed or examined as no test item-related morphologic changes were detected in group 4.

 

 The animals survived the scheduled treatment period.

 No clinical signs or alterations in food consumption, body weights, sensory activity, grip strength, locomotor activity, hematology, coagulation and biochemistry parameters were recorded attributed to the treatment. 

No treatment-related macroscopic or microscopic alterations or alterations in organ weights were observed.