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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 31, 2018 to August 21, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,​7-​Naphthalenedisulfoni​c acid, 5-​(acetylamino)​-​3-​[2-​[5-​[[4-​chloro-​6-​[[3-​[[2-​(sulfooxy)​ethyl]​sulfonyl]​phenyl]​amino]​-​1,​3,​5-​triazin-​2-​yl]​amino]​-​2-​sulfophenyl]​diazenyl]​-​4-​hydroxy-​, sodium salt (1:4)
EC Number:
695-779-2
Cas Number:
80019-35-8
Molecular formula:
C29H21ClN8O17S5.4Na
IUPAC Name:
2,​7-​Naphthalenedisulfoni​c acid, 5-​(acetylamino)​-​3-​[2-​[5-​[[4-​chloro-​6-​[[3-​[[2-​(sulfooxy)​ethyl]​sulfonyl]​phenyl]​amino]​-​1,​3,​5-​triazin-​2-​yl]​amino]​-​2-​sulfophenyl]​diazenyl]​-​4-​hydroxy-​, sodium salt (1:4)
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Animal Specifications:
- Source: National Institute of Biosciences, Pune, India
- Health Status: nulliparous and non-pregnant
- Body weight: 178~195g
- Age: 8-10 weeks at the time of dosing for 9 animals; 9-11 weeks at the time of dosing for 9 animals.
- Acclimatisation: Animal no. 01-03 were acclimatized for 9 days, 04-06 for 12 days; 07-09 for 15 days, 10-12 for 06 days prior to administration of the test item.

Husbandry Conditions:
- Diet: ad libitum
- Water: ad libitum
- Bedding: All cages were provided with corn cobs
- Husbandry: The animals were housed individually in polycarbonate cages.
- Room Sanitation: The experiment room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle: All the cages and water bottle were changed at least twice everyweek.

Experimental Room Condition:
- Temperature (°C): 20.20~23.60°C
- Humidity (%): 42.70~65.50%
- Photoperiod (hrs dark / hrs light): 12-hrs dark / 12-hrs light cycle
- Air changes: More than 12 changes per hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Doses:
2000 mg/kg b.w.
No. of animals per sex per dose:
For Dose Step 1: three females
For Dose Step 2: three females
Details on study design:
1. Starting Dose
Three animals were used for each step. The starting dose was selected as 300 mg/kg body weight as no information available with Pesticide Manual. The time interval between treatments was determined by the onset, duration and severity of toxic signs. Treatment of animals at the next dose was delayed until confidence of survival of the previously dosed animals is attained.

2. Dose Formulation
The test item was soluble in Distilled water. Hence, distilled water was selected as a vehicle. In step-I, 600 mg of test item was added to the vehicle (Distilled water) and mixed well. The formulation was then transferred to a measuring cylinder. The stock concentration of 30 mg/mL was achieved by making up the volume up to 20 mL in the measuring cylinder. In step-II, 300 mg of test item was added to the vehicle (Distilled water) and mixed well. The stock concentration of 30 mg/mL was achieved by making vehicle (Distilled water) and mixed well. The stock concentration of 200 mg/mL was achieved by making up the volume up to 10 mL in the Measuring cylinder. In step-III, 4000 mg of test item was added to the vehicle (Distilled water) and mixed well. The stock concentration of 200 mg/mL was achieved by making up the volume up to 20 mL in the measuring cylinder. In step IV, 2000 mg of test item was added to the vehicle (Distilled water) and mixed well. The stock concentration of 200 mg/mL was achieved by making up the volume up to 100 mL in the Measuring cylinder. Before dosing, the dose formulation was continuously mixed using magnetic stirrer to ensure the homogeneity. Two sets of dose formulation sample (2 mL of each layer) which includes (Top, Middle and Bottom Layer) collected from the dose formulation samples prepared in Step I and III was analysed for active ingredient content.

3. Test Item Dosing Procedure
The maximum dose volume administered was 10 mL/kg body weight. A total of twelve female Wistar rats were selected for acute oral toxicity study. The animals were fasted for approximately 17.5 to 18.0 hours prior to dosing and for approximately 3.0to 3.5 hours post dosing, feed was withheld but drinking water provided ad libitum. Initially, three rats of Group G1 (Step I) were administered orally at a dose of 300 mg/kg body weight; no mortality was observed in all three animals. Hence, another set of three rats of Group G1 (Step II were administered at 300 mg/kg body weight and no mortality was observed at this dose level.

Based on the results of Group G1, another set of three rats G2 (Step III) were administered orally at a dose of 2000 mg/kg body weight; no mortality was observed in all three rats. Hence another set of three rats of Group G2 (Step IV) were administered at 2000 mg/kg body weight and no mortality was observed at this dose level. Hence no further dosing was performed. The time internals between dosing were determined by the onset, duration and severity of toxic signs. Animal no. 01 to 03 were dosed between 11:28 to 11:31 a.m.; Animal no. 04 to 06 were dosed between 11:12 to 11:15 a.m. Animal no. 07 to 09 were dosed between 11:20 to 11:23 a.m. and Animal no. 10 to 12 were dosed between 11.37 to 11.38 a.m.. The interval period approximately 48 hours was maintained between step I and step II dosing.

Results and discussion

Preliminary study:
Initially, three rats of Group G1 (Step I) were administered orally at a dose of 300 mg/kg body weight; no mortality was observed in all three animals. Hence, another set of three rats of Group G1 (Step II were administered at 300 mg/kg body weight and no mortality was observed at this dose level.

Based on the results of Group G1, another set of three rats G2 (Step III) were administered orally at a dose of 2000 mg/kg body weight; no mortality was observed in all three rats. Hence another set of three rats of Group G2 (Step IV) were administered at 2000 mg/kg body weight and no mortality was observed at this dose level. Hence no further dosing was performed.
Effect levels
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
5 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed in all the animals.
Clinical signs:
other: At 300 mg/kg body weight Group G1 (Step I and II) all animals were normal at 30 min, 1 hour, 2 hours, 3 hour, 4 hours till sacrifice. No mortality was observed in all animals. At 2000 mg/kg body weight, all the animal group G2 (Step III and IV) were norma
Gross pathology:
No pathology abnormalities were observed on external and internal gross examination of terminally sacrificed animals.
Other findings:
Homogeneity and Active Ingredient Analysis Dose Formulation:
The active ingredient Concentrations of the test item in dose formulation was analyzed by validated HPLC method. About 2.0 mL of dose formulation (Top, Middle and Bottom Layer) with duplicate was collected (30 mg/mL) from G1 (Step I) and G2 (step III) (200 mg/mL) and analyzed for active ingredient content, The percentage of mean active ingredient content obtained at 30 mg/mL and at 200 mg/mL concentration is for CR SR94. The analysis result showed that the analyzed content on CR SR94 was within the acceptable limit (85-115% with %RSD should be <=10%).

Any other information on results incl. tables

Table 1. Individual Animal Body Weight (g) and Body Weight Changes (%)

Animal No. Group/
Dose (mg/kg)/Step
Dose Volume (mL)* Body Weight (gram) Body Weight Change (%)
Day 0 Day 7 Day 14 Found Dead Day 0-7 Day 0-14
01 G1/
300/Step I
2.0 195 207 220 ./. 6.15 12.82
02 1.9 187 199 212 ./. 6.42 13.37
03 1.8 178 190 203 ./. 6.74 14.04
04 G1/
300/Step II
1.9 185 197 209 ./. 6.49 12.97
05 1.8 182 195 207 ./. 7.14 13.74
06 1.8 184 198 210 ./. 7.61 14.13
07 G2/
2000/Step III
1.9 185 197 209 ./. 6.49 12.97
08 1.8 184 197 210 ./. 7.07 14.13
09 1.8 181 193 206 ./. 6.63 13.81
10 G2/
2000/Step IV
1.8 184 197 209 ./. 7.07 13.59
11 1.8 183 195 208 ./. 6.56 13.66
12 1.8 183 194 207 ./. 6.01 13.11

*Dose volume calculated based on day 0 boy weight; ./. = Not applicable

Table 2. Summary of Animal Body Weight (g) and Body Weight Changes (%)

Group/Dose (mg/kg)/Step Rats Body Weight (g) Body Weight Change (%)
Day 0 Day 7 Day 14 Day 0-7 Day 0-14
G1/
300/Step I
Mean 186.67 198.67 211.67 6.44 13.41
SD 8.50 8.50 8.50 0.30 0.61
n 3 3 3 3 3
G1/
300/Step II
Mean 183.67 196.67 208.67 7.08 13.61
SD 1.53 1.53 1.53 0.56 0.59
n 3 3 3 3 3
G2/
2000/Step III
Mean 183.33 195.67 208.33 6.73 13.64
SD 2.08 2.31 2.08 0.30 0.60
n 3 3 3 3 3
G2/
2000/Step IV
Mean 183.33 195.33 208.00 6.55 13.45
SD 0.58 1.53 1.00 0.53 0.30
n 3 3 3 3 3

SD = Standard Deviation, n = Number of Animals

Table 3. Individual Animal Clinical Signs and Symptoms

Animal No. Group/
Dose (mg/kg)/
Step
Hours (Day 0)
1/2 1 2 3 4
01

G1/300/

Step I

1 1 1 1 1
02 1 1 1 1 1
03 1 1 1 1 1
04

G1/300/

Step II

1 1 1 1 1
05 1 1 1 1 1
06 1 1 1 1 1
07

G2/2000/

Step III

1 1 1 1 1
08 1 1 1 1 1
09 1 1 1 1 1
10

G2/2000/

Step IV

1 1 1 1 1
11 1 1 1 1 1
12 1 1 1 1 1

Table 3. Individual Animal Clinical Signs and Symptoms (continued)

Animal No. Group/
Dose (mg/kg)/
Step
Days after post dosing (Morning)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
01 G1/300/Step I 1 1 1 1 1 1 1 1 1 1 1 1 1 1
02 1 1 1 1 1 1 1 1 1 1 1 1 1 1
03 1 1 1 1 1 1 1 1 1 1 1 1 1 1
04 G1/300/Step II 1 1 1 1 1 1 1 1 1 1 1 1 1 1
05 1 1 1 1 1 1 1 1 1 1 1 1 1 1
06 1 1 1 1 1 1 1 1 1 1 1 1 1 1
07 G2/2000/Step III 1 1 1 1 1 1 1 1 1 1 1 1 1 1
08 1 1 1 1 1 1 1 1 1 1 1 1 1 1
09 1 1 1 1 1 1 1 1 1 1 1 1 1 1
10 G2/2000/Step IV 1 1 1 1 1 1 1 1 1 1 1 1 1 1
11 1 1 1 1 1 1 1 1 1 1 1 1 1 1
12 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Table 3. Individual Animal Clinical Signs and Symptoms (continued)

Animal No. Group/
Dose (mg/kg)/
Step
Days after post dosing (Evening)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
01 G1/300/Step I 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
02 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
03 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
04 G1/300/Step II 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
05 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
06 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
07 G2/2000/Step III 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
08 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
09 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
10 G2/2000/Step IV 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
11 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.
12 1 1 1 1 1 1 1 1 1 1 1 1 1 ./.

Table 4. Individual Animal Mortality Record

Animal No. Group/
Dose (mg/kg)/
Step
Day of Observation (Day 0 to 14)
Morning Observation Evening Observation
01 G1/300/Step I No mortality and norbidity No mortality and norbidity
02 No mortality and norbidity No mortality and norbidity
03 No mortality and norbidity No mortality and norbidity
04 G1/300/Step II No mortality and norbidity No mortality and norbidity
05 No mortality and norbidity No mortality and norbidity
06 No mortality and norbidity No mortality and norbidity
07 G2/2000/Step III No mortality and norbidity No mortality and norbidity
08 No mortality and norbidity No mortality and norbidity
09 No mortality and norbidity No mortality and norbidity
10 G2/2000/Step IV No mortality and norbidity No mortality and norbidity
11 No mortality and norbidity No mortality and norbidity
12 No mortality and norbidity No mortality and norbidity

Table 5. Gross Necropsy Observation and Microscopic Observation

Animal No. Group/
Dose (mg/kg)/
Step
Mode of Death Gross Observation Microscopic Observation
External  Internal
01 G1/300/Step I TS NAD NAD ./.
02 TS NAD NAD ./.
03 TS NAD NAD ./.
04 G1/300/Step II TS NAD NAD ./.
05 TS NAD NAD ./.
06 TS NAD NAD ./.
07 G2/2000/Step III TS NAD NAD ./.
08 TS NAD NAD ./.
09 TS NAD NAD ./.
10 G2/2000/Step IV TS NAD NAD ./.
11 TS NAD NAD ./.
12 TS NAD NAD ./.

TS = Terminal Sacrifice; NAD = No abnormality detected.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this acute oral toxicity study in female Wistar Rats, the acute oral LD50 cut-off value of CR SR94 was found to be greater than 2000 mg/kg body weight and classified as under with the cut-off value at 5000 mg/kg body weight.
The Globally Harmonized System of Classification and Labelling of Chemicals (GHS): "Category 5" or "Unclassified".
Executive summary:

This study was performed as per OECD423 guideline. A total of twelve female Wistar Rats were selected for acute oral toxicity study. The animals were fasted for 17.5 to 18.0 hours prior to dosing and for 3.0 to 3.5 hours post dosing, feed was withheld but drinking water provided ad libitum. The time intervals between doing were determined by the onset, duration and severity of toxic signs.

 

Initially, three rats of Group G1 (Step I) were administered orally at a dose of 300 mg/kg body weight; no mortality was observed in all the three animals. Hence, another set of three rats of Group G1 (Step II) were administered at 300 mg/kg body weight and no mortality was observed at this dose level.

 

Based on the results of Group G1, another set of three rats G2 (Step III) were administered orally at a dose of 2000 mg/kg body weight; no mortality was observed in all the three rats. Hence, further confirmatory dosing was performed with 2000mg/kg body weight as per dosing criteria.

 

Body weights were recorded on day 0 (prior to dosing) and on days 7 and 14. The mean body weight of all the animals of Group G1 (Step I and II) and G2 (Step III and IV) treated at 300mg/kg body weight and 2000mg/kg body weight respectively showed gain on day 7 and 14, as compared to day 0 body weight.

 

At 300mg/kg body weight Group G1 (Step I and II) all animals were normal at 30 min, 1 hour, 2 hours, 3 hours, 4hours, till sacrifice. No mortality was observed in all animals.

 

At 2000mg/kg body weight, all the animals group G2 (Step III and IV) were normal at 30 min, 1 hour, 2 hours, 3 hours, 4hours observation. No mortality was observed in all animals.

 

The active ingredient concentrations of the test item in dose formulation was analyzed by validated HPLC method. About 2.0 mL of dose formulation (Top, Middle and Bottom Layer) with duplicate was collected (30 mg/mL) from G1 (Step I) and G2 (Step III) (200 mg/mL) and analyzed for active ingredient content. The percentage of mean active ingredient content obtained at 30 mg/mL and at 200 mg/mL concentration is for CR SR94. The analysis result showed that the analyzed content of CR SR94 was within the acceptable limit (85-115% with %RSD should be <=10%).

 

The Mean active ingredient content of CR SR94 on dose formulation analysis: 

 Dose (mg/kg body weight)  Weight of the test item taken (mg)  Volume made upto (mL)  Calculated concentration (mg/mL)  Expected concentration CR SR94 (mg/L) on purity basis Analyzed mean content of CR SR94 (mg/L)  Analyzed content of CR SR94 in % basis 
 300 600  20  30  120.150  127.348  105.99 
2000  4000  20  200  160.200  162.453  101.41 

No pathological abnormalities were observed on external and internal gross examination of terminally sacrificed animals.

 

Under the conditions of this acute oral toxicity study in female Wistar Rats, the acute oral LD50 cut-off value of CR SR94 was found to be greater than 2000 mg/kg body weight and classified as under with the cut-off value at 5000 mg/kg body weight and classified as under.

 

The Globally Harmonized System of Classification and Labelling of Chemicals (GHS): "Category 5" or “Unclassified".