Bisguanidinium phosphate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-15 to 2019-02-18

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI (21.04.2016)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bisguanidinium phosphate

EC Number:

226-552-4

EC Name:

Bisguanidinium phosphate

Cas Number:

5423-23-4

Molecular formula:

CH5N3.1/2H3O4P

IUPAC Name:

Diguanidinium hydrogen phosphate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han: WIST

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TOXI COOP ZRT., Cserkesz u. 90. 1103 Budapest, Hungary
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rat, 8 weeks old in first, second and third step
- Weight at study initiation: 164 - 165 g (first step), 167 - 173 g (second step), 165 - 173 g (third step)
- Fasting period before study: The day before treatment the animals were fasted. The food but not water was withheld overnight. Food was given back 3 hours after the treatment
- Housing: Group caging (3 animals/cage); cage type: Type III polypropylene/polycarbonate, laboratory bedding
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water (e.g. ad libitum): Animals received tap water from municipal supply, as for human consumption from bottle ad libitum
- Acclimation period: 5 days in first step, 6 days in second step and 7 days in third step

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): above 10 air exchanges/hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 17 Jan 2019 To: 06 Feb 2019 (first group), 07 Feb 2019 (second group), 24 and 25 Jan 2019 (third group)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Remarks:

Helianthi annui oleum raffinatum

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 300 and 2000 mg/kg bw
- Amount of vehicle (if gavage): 10 mL/kg bw
- Justification for choice of vehicle:
- Lot/batch no. (if required): 1809-4563

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION (if unusual): All doses were formulated in the vehicle. Concentration of formulations was adjusted to maintain a treatment volume of 10 mL/kg bw. The test item was applied in a concentration of 30 and 200 mg/mL. Formulations were prepared just before the administration and were stirred continuously during the treatment.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Starting dose was selected on the basis of sponsor’s request based on data/information at hand for a similar substance.

Doses:

300, 2000 mg/kg bw

No. of animals per sex per dose:

6 (300 mg/kg bw), 3 (2000 mg/kg bw)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Body weight measured on day 0, 7 and 15, Clinical observations at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h after the treatment, and once a day for 14 days thereafter
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, gross pathology

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

500 mg/kg bw

Based on:

test mat.

Mortality:

No death occurred at 300 mg/kg bw single oral dose of the test item Bisguanidinium phosphate. All female rats in step 1 and step 2 survived until the end of the 14-day observation period.
All rats dosed at 2000 mg/kg bw (step 3) died during the study. Animal No.: 3578 died on the treatment day, 3 hours after the treatment. Two animals (No.: 3577, 3579) died on Day 1. The deaths seemed to be consequences of systemic toxic effect of the test item.

Clinical signs:

In group 1 and 2 treated with 300 mg/kg bw no treatment related symptoms were observed throughout the 14-day post-treatment period.
In group 3 treated with 2000 mg/kg bw dose clinical signs of reaction comprised of decreased activity (4 cases of 13 observations), tremor (4/13), abnormal gait (4/13), closed eyes (2/13) and piloerection (4/13). These symptoms were observed in two animals (No.: 3577, 3579). The scores of symptoms were as follows: decreased activity (-1), tremor (+2), abnormal gait (+2), closed eyes (+1) and piloerection (+1). These symptoms were observed on the treatment day between 3 and 4 hours after the treatment and were related to the toxic effect of the test item. The clinical symptoms with pathological changes refer to potential neurotoxic effect of test item.

Body weight:

The mean body weight of animals treated with 300 mg/kg bw corresponded to their species and age throughout the study.
The mean body weight and body weight gain data of group 3 (2000 mg/kg bw) could not be evaluated, because of mortalities.

Gross pathology:

Six animals treated with 300 mg/kg bw dose survived until the scheduled necropsy on Day 15.
Three animals treated with 2000 mg/kg bw dose spontaneously died during the study. One animal (No.: 3578) was necropsied on the treatment day and two animals (No.: 3577, 3579) were necropsied on Day 1.
Slight hydrometra was found in animal No.: 3574 of group 1 and moderate hydrometra was detected in animal No.: 3576 of group 1 and in animal No.: 3585 of group 2, as well. Hydrometra is physiological finding and connected to the oestrus cycle of the animal. No pathological changes were found indicate a toxic effect of the test item during the macroscopic examination of animals treated with 300 mg/kg bw dose.
An external necropsy finding as frothy discharge around the mouth was found in all animals treated with 2000 mg/kg bw and frothy discharge around the nose was detected in two animals (No.: 3577, 3579). Besides, internal necropsy findings were found in same group. Stomach was full of gas in all animals and was hyperaemic in animal No.: 3579. Light coloured spleen was recorded in all animals. Liver was dark coloured in animal No: 3578. Hyperaemic intestines were observed in animal No.: 3578. Intestines were full of gas in animal No.: 3579.

Any other information on results incl. tables

Summary of Lethality

Groups	Treatment		Lethality
	Test Item	Dose (mg/kg bw)	Females
1	Bisguanidinium phosphate Step 1	300	0/3
2	Bisguanidinium phosphate Step 2	300	0/3
3	Bisguanidinium phosphate Step 3	2000	3/3

Summary of gross pathology findings

Observations Females	Bisguanidinium phosphate
	300 mg/kg bw	2000 mg/kg bw
Around the mouth: frothy disharge	0/6	3/3
Around the nose: frothy disharge	0/6	2/3
Stomach: full of gas	0/6	3/3
hyperaemic	0/6	1/3
Spleen: light coloured	0/6	3/3
Liver: dark coloured	0/6	1/3
Intestines: hyperaemic	0/6	1/3
full of gas	0/6	1/3
Hydrometra	3/6	0/3
Normal	3/6	0/3

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

No death occurred after the single 300 mg/kg bw oral dose of Bisguanidinium phosphate. All out of three animals died in group 3 treated with 2000 mg/kg bw dose.
There were no toxic clinical signs and any test item related effect found in body weights and body weight gains in 300 mg/kg bw dose during the study. The all observed clinical signs were related to the effect of the test item in 2000 mg/kg bw dose. Autopsy revealed no treatment related pathological changes in 300 mg/kg bw dose.
The external and internal necropsy findings observed in 2000 mg/kg bw dose were related to the effect of the test item.
The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423: LD50 = 500 mg/kg bw, GHS category 4

Executive summary:

The Acute Oral Toxicity Study (Acute Toxic Class Method) of Test Item Bisguanidinium phosphate in rats was conducted following OECD guideline 423 under GLP compliance.

Starting dose was selected on the basis of sponsor’s request based on data/information at hand for a similar substance.

The acute toxic class method was carried out involving a stepwise procedure with the use of 300 mg/kg bw as the starting dose in three female rats. No animal died in the first group, so further three female rats were treated with the same (300 mg/kg bw) dose. No animal died in the second group, too. Based on the testing scheme further three female rats were treated with the 2000 mg/kg bw dose. All animals died in this group, so the test was finished as the stopping criteria of Annex 2c of OECD Guideline No. 423 were met.

Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals died on the treatment day or on Day 1, as well as 15th day after the treatment in survivor animals.

Lethality, Clinical symptoms and Body weight

All female rats in step 1 and step 2 survived until the end of the 14-day observation period.

One rat dosed at 2000 mg/kg bw Bisguanidinium phosphate died on the treatment day 3 hours after the treatment and two rats of same group died on Day 1. In the first and second step, no clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the general state and behaviour of experimental animals were normal.

In third step, Clinical - and emotion symptoms (decreased activity, tremor, closed eyes), disturbance of coordination (abnormal gait) and disturbance of the autonomic functions (piloerection) were observed in two animals on the treatment day between 3 and 4 hours after the treatment.

The body weight development was undisturbed in all survivor animals.

Gross pathology

All animals treated with 300 mg/kg bw dose survived until the scheduled autopsy on Day 15. All of the three animals treated with 2000 mg/kg bw dose died spontaneously during the study and were necropsied on the treatment day or on Day 1. External necropsy finding as frothy discharge around the mouth and/or nose was observed in animals treated with 2000 mg/kg bw dose. Internal necropsy findings were recorded in 2000 mg/kg bw dose group as stomach full of gas or was hyperaemic, light coloured spleen, dark coloured liver, hyperaemic intestines and intestines full of gas.

All organs of the animals treated with 300 mg/kg bw proved to be free of treatment related gross pathological changes.

The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 as below:

Dose (mg/kg bw)	Mortality (dead/treated)	LD50 (mg/kg bw)	GHS category
300 2000	0/6 3/3	500	4