Bisguanidinium phosphate

Endpoint summary

Description of key information

Acute toxicity:oral:

Kuthy, 2019: Key study, OECD TG 423, Acute Toxic Class method, rat, LD50 = 500 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

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

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

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)

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

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.

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

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Quality of whole database:

High quality due to guideline study and supporting robust read across.

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute Toxicity: oral

Key study - Kuthy, 2019

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 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

supporting - Read across: WIL Research Europe, 2013

An acute oral toxicity study was performed with rats according to OECD/EC test guidelines and GLP principles following the acute toxic class method. Three rats were exposed to 2000 mg/kg bw guanidinium phosphate (1:1), all three died on day 1. Lethargy, tremor, abnormal posture, flat and/or hunched posture, abnormal gait, piloerection, restless and fearful behavior and/or chromodacryorrhoea were noted on the day of death. Macroscopic post mortem examination of these animals revealed in two animals several reddish foci in the glandular mucosa of the stomach and/or gastro-intestinal tract distended with gas.

Two groups of three rats were exposed to 300 mg/kg bw, no further mortality occurred. Three animals showed hunched posture and piloerection on Day 1, no abnormalities were found at necropsy. The body weight gain shown by the surviving animals over the study period was considered to be similar to that expected of normal untreated animals of the same age and strain.

Based on these data, the LD50 for guanidinium phosphate (1:1) was determined to be between 300 and 2000 mg/kg bw. These LD50 values were calculated for the target substance accounting for the higher content of guanidinium ion. As a result, the LD50 value for bisguanidinium phosphate ranges between 206 and 1376 mg/kg bw.

The similar results of the source and the target substance for acute toxicity underline the similar toxicological behaviour. This demonstrates that the read-across is justified and tends to be overconservative.

Justification for classification or non-classification

Based of the oral toxicity study with the test item bisguanidinium phosphate an LD₅₀ of 500 mg/kg bw was determined. Thus, the substance is classified according to Regulation (EC) No 1272/2008 for Acute Oral Toxicity Category 4.