Guanidinium nitrate

Endpoint summary

Description of key information

Acute toxicity data on Guanidine Nitrate are available for the oral, inhalation and dermal route.

The data available from three studies for the oral route all indicate LD50 values in the range between 300 and 2000 mg/kg body weight. For the dermal route, the LD50 is > 2000 mg/kg body weight. In an inhalation study it was shown that at the highest technically achievable dose of 0.853 mg/L air no mortalities occurred. Additional data from an acute inhalation toxicity study with the read-across substance guanidine hydrochloride are available, with a LC50 value for females of 3.181 mg/L air.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-02-29 to 1984-04-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Health effects test guidelines, August 1982, EPA 560/6-82-001

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin-Kingman Inc., Fremont, CA
- Age at study initiation: Males: 58 days at dosing - Females 33 to 41 days at dosing
- Weight at study initiation: Males 190 to 225 g at dosing - Females 140 to 174 g at dosing
- Fasting period before study: Males: not stated - Females: overnight
- Housing: individually in stainless steel wire mesh cages with automatically flushing dumptanks
- Diet: ad libitum: Certified Purina Rodent Chow No. 5002 (Ralston Purina Company, Checkerboard Square St. Louis, MD)
- Water: ad libitum: automatic lick dispensers
- Acclimation period: quarantine: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): different rooms: (22.8 to 25.0) or (20.6 to 22.8) or (22.2 to 26.7 with a temporary drop to 18.9)
- Humidity (%): different rooms: (44 to 52 with a temporary spike to 64) or (44 to 64 with a temporary spike to 74 or (32 to 44)
- Air changes (per hr): not described
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: Males from 1984-03-14 to 1984-03-28 - Females from: 1984-03-21 to: 1984-04-11

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

Tween® 80 + sterile water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0,2 % methylcellulose, 0,4 % Tween® 80 in sterile water for injection
- Amount of vehicle (if gavage): Males 1.9 to 2.6 ml - Females 1.2 to 1.8 ml for dosed animals
2.0 ml for all vehicle control group animals
- Justification for choice of vehicle: data not available
- Lot/batch no. (if required):
methylcellulose (Sigma, St. Louis, MO, Lot Number 72F-047B)
Tween® 80 (Fisher Scientific, Fairtown, NJ, Lot Number 713137)
sterile water for injection (Cutter Medical, Berkeley, CA, Lot Number 426-27, 13 Mar 84, Lot Number CH7936, 20 Mar 1984)
- Purity: data not available

MAXIMUM DOSE VOLUME APPLIED: 2.6 ml

DOSAGE PREPARATION (if unusual):

Depending upon the dose level, various amounts of Guanidine Nitrate were weighed in a Mettler AK 160 Electronic Balance and were added to 50 ml of the vehicle for males and 25 ml of the vehicle for females to yield the desired dosing concentration. Complete analyses of the dosing
solutions/suspensions was performed for homogeneity and verification of concentrations.

Doses:

Males: 311 - 826 - 1000 - 1210 - 1470 mg/kg bw
Females: 610 - 718 - 847 - 1000 - 1180 - 1390 mg/kg bw

No. of animals per sex per dose:

Total animals dosed: 108
Dosing groups: 10 animals per sex
Exception: Females lowest dose group: 8 animals

Control animals:

yes

Remarks:

vehicle: 5 Males + 5 Females

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: daily
- Frequency of weighing: weekly
- Necropsy of survivors performed: yes
- Other examinations performed: (1) clinical signs (2) body weight (3) histopathology
Details for clinical signs:
1.1 behaviour ( includes ataxia, disorientation, hyperactivity, jumping, irritableness, inactivity, changes in preening, prostrate condition,
aggressiveness, sonnolence, tremors, twitching)
1.2 gastrointestinal (includes increased salivation, material in mouth, diarrhea, perianal staining)
1.3 respiratory (includes reddish nasal discharge and stains on head, increase in respiratory rate, decrease in respiratory depth)
1.4 ocular (Includes reddish material around eyes (chromodacryorrhea), conjunctivitis)
1.5 rough coat
1.6 hunched posture
1.7 normal throughout

Statistics:

Statistical analyses were performed on the study results. Selected lethal dose values were derived by probit analysis according to the maximum likelihood method. The program PROBIT, developed for the Data General Computer, Model MV8000, was used to determine the probit curve and lethal dose values.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

989.6 mg/kg bw

95% CL:

> 793 - < 1 138

Remarks on result:

other: LD50 = 989.6 +/- 68.7

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

729.8 mg/kg bw

95% CL:

> 641 - < 799

Remarks on result:

other: LD50= 729.8 +/- 34.3

Mortality:

Forty-six (74 %) of the deaths occurred between 1 and 8 hours after dosing. An additional 13 (21 %} deaths occurred between 8 and 24 hours after dosing. Three animals were found dead on the morning of the second day (between 36 and 47 hours after dosing). Furthermore, 2 males and 1 female were inappropriately dosed resulting in latrogenic deaths. The remaining rats survived until completion of the study.

Clinical signs:

other: Guanidine nitrate produced clinical signs at each dose level. The most frequently observed signs were behavioral (63 of 99 animals dosed with test compound), gastrointestinal (GI) tract symptoms (37 of 99), and respiratory (26 of 99) signs. Most clinical

Gross pathology:

Latrogenic deaths due to oesophagal perforatin or tracheal tears and deposition of the test substance in the thorax occured in 2 males and 1 female. Thirteen females had multiple red foci on the thymus. None of the males had this lesion.

Other findings:

- Organ weights: not determined
- Histopathology: The presence of multiple red foci in the thymuses of the females was the only gross lesion which was attributable to the test compound. The presence of foci exhibited a dose-response relationship. The remainng lesions were considered incidential findings which are not compound related. No lesions were seen in the vehicle control animals.

Lethal dose values were calculated by probit analysis, and the equation for the probit regression line was:

Y = -19.6 + 8.2 log X for males and Y = -32.5 + 13.1 log X for females, where X is the dose and Y the corresponding probit value.

Misdosed animals were excluded from statistical analysis and eliminated from the study.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 values for Guanidine Nitrate were determined to be 989.6 +/- 68.7 mg/kg in male and 729.8 +/-34.3 mg/kg in female animals.
Guanidine Nitrate is classified with Hazard Category 4 for acute oral toxicity (harmful if swallowed).

Executive summary:

In an acute oral toxicity study according to the Health effects test guidelines, August 1982, EPA 560/6-82-001, groups of Sprague Dawley rats (10/sex) were given a single oral dose of  guanidinte Nitrate (99.99 % a.i.) in methylcellulose, Tween® 80, sterile water at doses of 311, 826, 1000, 1210 and 1470 mg/kg bw (males) and 610, 718, 847, 1000, 1180 and 1390 mg/kg bw (females) and observed for 14 days.

 

Oral LD50   Males = 989.6 ± 68.7 mg/kg bw (95% C.I. > 793 - < 1138)

                      Females = 729.8 ± 34.3 mg/kg bw (95% C.I. > 641 - < 799)

     

 

65 animals died during the study, most of them during the first 8 hours after dosing. Weight gains of surviving animals were not significantly affected by dosing.

 

The primary category of clinical signs was behavioral (e.g., inactive, irritable, disoriented, hyperactive, ataxic) which was observed in 63 of 99 animals dosed with Guanidine Nitrate. Other categories of frequently observed clinical signs were gastrointestinal (e.g., material in mouth, perlanal staining, increased salivation, diarrhea) which was observed in 37 of 99 animals, and respiratory (e.g. reddish nasal discharge, increased rate and/or decreased depth of respiration) which was observed in 26 of 99 animals. These findings suggest that guanidine nitrate exhibits a primary effect on the central nervous / neuromuscular system.

The presence of multiple red foci in the thymuses of the females was dose related and the only gross lesion which was attributable to the test compound.

 

According to CLP, EU GHS (Regulation (EC) No 1272/2008), Guanidine Nitrate is classified with Hazard Category 4 for acute oral toxicity (harmful if swallowed). Findings may suggest that the guanidine ion exhibits a primary effect on the central nervous / neuromuscular system. However, effects were observed in the range of classification Hazard Category 4, therefore on the basis of available data no additional classification for STOT-SE is considered necessary. A sub-chronic repeated dose toxicity study is proposed, which will provide additional more detailed information for evaluation.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Supporting information for read-across, structural analogue approach.

Qualifier:

according to guideline

Guideline:

other: Health Effects Testing Guidelines. August 1982; EPA 560/6-82-001

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

no

Species:

mouse

Strain:

ICR

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The weight of base equivalent was used to calculate the required dose.

Doses:

0, 398, 495, 501, 631, 794 mg Guanidine/kg bw (males), corresponding to 0, 648.74, 806,85, 816.63, 1028.53, 1294.22 mg/kg bw as Guanidine hydrochloride
0, 316, 398, 501, 631, 794 mg Guanidine/kg bw (females), corresponding to 0, 515.08, 648.74, 816.63, 1028.53, 1294.22 mg/kg bw as Guanidine hydrochloride

No. of animals per sex per dose:

5 (vehicle control), 10 (treated groups)

Control animals:

yes

Statistics:

Probit analysis

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

570.8 mg/kg bw

Based on:

other: Guanidine

95% CL:

>= 491 - <= 694

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

621 mg/kg bw

Based on:

other: Guanidine

95% CL:

>= 492 - <= 1 183

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

930.4 mg/kg bw

Based on:

test mat.

Remarks:

Guanidine hydrochloride

95% CL:

>= 800.33 - <= 1 131.22

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

997.56 mg/kg bw

Based on:

test mat.

Remarks:

Guanidine hydrochloride

95% CL:

>= 801.96 - <= 1 928.29

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The oral LD50 for Guanidine base in mice was 570.8 mg/kg bw (95% c.i. 491 - 694 mg/kg bw) in males and 612 mg/kg bw (95% c.i. 492 – 1183 mg/kg bw) in females, corresponding to 930.4 mg/kg bw (95% c.i. 800.33 - 1131.22 mg/kg bw) in males and 997.56 mg/kg bw (95% c.i. 801.96 - 1928.29 mg/kg bw) in females expressed as Guanidine hydrochloride.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Supporting information for read-across, structural analogue approach.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Remarks:

study conducted before implementation of GLP

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

other: ClVO colony (Wistar derived)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Doses:

800, 1000, 1200, 1400 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Statistics:

Litchfield and Wilcoxon

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

1 120 mg/kg bw

Based on:

test mat.

95% CL:

>= 991 - <= 1 266

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The oral LD50 for Guanidine hydrochloride was 1120 mg/kg bw (95% c.i. 991 – 1266 mg/kg bw).

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984-01-19 to 1984-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Supporting information for read-across, structural analogue approach.

Qualifier:

according to guideline

Guideline:

other: EPA TS-792 Acute exposure, oral toxicity. Health effects test guidelines, EPA, August 1982; EPA 560/6-82-001

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin Kingman, Fremont, CA
- Weight at study initiation: 150 – 255 g
- Fasting period before study: yes, over night
- Housing: individually
- Diet (e.g. ad libitum): Certified Purina Rodent Chow Diet 5062, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 11 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.2°C to 25°C
- Humidity (%): 40 to 53%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 0.74-1.02 mL/animal in males, 0.59-0.74 mL/animal in females
- Justification for choice of vehicle: test substance is soluble in water

Doses:

0, 278, 360, 464, 600, 775 mg/kg bw (expressed as active ingredient Guanidine), corresponding to 0, 453.14, 756.32, 978, 1263.25 mg/kg bw Guanidine hydrochloride

No. of animals per sex per dose:

9 in treated groups; 5 in vehicle control and cage control

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Clinical signs, mortality: after 2 and 4 h, daily thereafter
Body weight: twice weekly
- Necropsy of survivors performed: yes; completet gross necropsy
- Other examinations performed: histopathology of CNS for animals with neurological symptoms

Statistics:

Probit analysis

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

556.5 mg/kg bw

Based on:

other: Guanidine

95% CL:

>= 492.3 - <= 630.9

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

474.6 mg/kg bw

Based on:

other: Guanidine

95% CL:

>= 402 - <= 562.7

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

907.1 mg/kg bw

Based on:

test mat.

Remarks:

Guanidine hydrochloride

95% CL:

>= 802.45 - <= 1 028.4

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

773.6 mg/kg bw

Based on:

test mat.

Remarks:

Guanidine hydrochloride

95% CL:

>= 655.3 - <= 917.2

Mortality:

- vehicle control: 0/5 males, 0/5 females died
- cage control: 0/5 males, 0/5 females died
- 278 mg/kg bw: 0/9 males, 0/8 females died
- 360 mg/kg bw: 0/9 males, 2/8 females died
- 464 mg/kg bw: 2/9 males, 5/9 females died
- 600 mg/kg bw: 5/9 males, 4/7 females died
- 775 mg/kg bw: 9/9 males, 9/9 females died
(reduced total numbers are due to misdosing; those animals were removed from the study)

Clinical signs:

other: most frequent clinical signs: - central nervous system-neuromuscular disturbances at all dose groups( 80 of 86 animals) - increased startle reflex, hyperactivity, and disorientation up to 464 mg/kg bw, recovery within one week - in the two higher dose gr

Gross pathology:

- no test compound-related lesions in the CNS (gross and light microscopic examination)
- the only test compound-related findings were in the gastrointestinal tract (black/serosanguinous material in the stomach, focal depression in the glandular mucosa of the stomach, black and diffuse red areas in the glandular mucosa of the stomach, black and tarry material in the intestine, black and tarry feces)
- in surviving animals no test compound-related gross lesions were reported; 2 male and 1 female rats had a unilateral dilated renal pelvis, which is considered incidential

Other findings:

- Histopathology:
Several rats exhibited central nervous system (CNS) signs. Some rats that exhibited CNS signs died and some others survived. The cerebrum, brainstem and cerebellum of 4 animals died were examined by light microscopy and no lesions were observed.

Sex       Endpoint       Effect level                                     95% CL

male       LD10       441.8 mg/kg bw (Guanidine base) 326.3 — 497.9

female       LD10       321.4 mg/kg bw (Guanidine base) 209.1 — 383.9

male       LD90       701.1 mg/kg bw (Guanidine base) 621.1 — 956

female       LD90       700.8 mg/kg bw (Guanidine base) 584.8 — 1089.9

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The oral LD50 for Guanidine hydrochloride in rats was 907.1 mg/kg bw (95% c.i. 802.45 - 1028.4) in males and 773.6 mg/kg bw (95% c.i 655.3 - 917.2) in females. Thus, Guanidine hydrochloride has to be classified for Acute oral toxicity, Category 4.

Executive summary:

In an acute oral toxicity study similar to OECD guideline 401, groups of fasted Sprague-Dawley rats (9/sex) were given a single oral dose of Guanidine hydrochloride (>98% purity, 61.8% base equivalent Guanidine) in water at doses of 0, 278, 360, 464, 600 and 775 mg/kg bw (expressed as Guanidine base), corresponding to 0, 453.14, 756.32, 978, 1263.25 mg/kg bw Guanidine hydrochloride and observed for 14 days.

No mortality occurred in the lowest dose group. 0/9 males, 2/8 females in the 360 mg/kg bw dose group died; 2/9 males, 5/9 females in the 464 mg/kg bw dose group; 5/9 males, 4/7 females in the 600 mg/kg bw dose group and 9/9 males, 9/9 females in the 775 mg/kg bw dose group. Reduced total numbers are due to misdosing; those animals were removed from the study.

Clinical signs were observed in the CNS (increased startle reflex, hyperactivity, and disorientation) and in the gastro-intestinal tract (hunched posture, increased salivation with some staining of the muzzle, and diarrhea) in all dose groups. However, there were no microscopic findings in cerebrum, brainstem and cerebellum of animals with CNS symptoms.

Gross pathology revealed black/serosanguinous material in the stomach, focal depression in the glandular mucosa of the stomach, black and diffuse red areas in the glandular mucosa of the stomach, black and tarry material in the intestine, black and tarry feces.

In the surviving animals no test compound-related gross lesions were reported.

Several rats exhibited central nervous system (CNS) signs. Some rats that exhibited CNS signs died and some others survived. The cerebrum, brainstem and cerebellum of 4 animals died were examined by light microscopy and no lesions were observed.

Oral LD50 Males = 556.5 mg Guanidine/kg bw (95% C.I. 492.3 - 630.9 mg/kg bw)

Oral LD50 Females = 474.6 mg Guanidine/kg bw (95% C.I. 402 - 562.7 mg/kg bw)

corresponding to:

Oral LD50 males = 907.1 mg/kg bw (95% c.i. 802.45 - 1028.4) Guanidine hydrochloride

Oral LD50 females = 773.6 mg/kg bw (95% c.i 655.3 - 917.2) Guanidine hydrochloride

Thus, Based on these data, Guanidine hydrochloride has to be classified for Acute toxicity, Category 4.

Reference 5

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984-03-14 to 1984-04-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Health effects test guidelines, August 1982, EPA 560/6-82-001

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., P.O. Box 29176, Indianapolis, IN 46229
- Age at study initiation: 60, 61 or 67 days at dosing
- Weight at study initiation: 25 g to 39 g
- Fasting period before study: 2 to 4 hours
- Housing: individually in stainless steel wire mesh cages in racks equipped with automatically flushing dumptanks
- Diet: ad libitum: Certified Purina Rodent Chow No. 5002 (Ralston Purina Company, Checkerboard Square St. Louis, MO)
- Water: ad libitum: provided by lixit valves on a central line
- Acclimation period: quarantine: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 to 23.3
- Humidity (%): 44 to 72 with occasional spikes to 80
- Air changes (per hr): not described
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1984-03-27 to 1984-04-17

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

polyoxyethylene-(20)-sorbitan monooleate Tween®80, sterile water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0.2 % methylcellulose, 0.4 % polyoxyethylene-(20)-sorbitan monooleate Tween®80 (MC-TW80) in sterile water for injection
- Amount of vehicle (if gavage): 0.21 to 0.27 ml in males - 0.17 to 0.24 ml in females - 0.2 ml in vehicle control group
- Lot/batch no. (if required):
methylcellulose (lot 12F-0478, expiration date, December 1986) was obtained from Sigma Chemical Company, St Louis, MO
Tween®80 (lot 713137, expiration date, December 1986) was obtained from Fisher Scientific Company, Chemical
The sterile water for injection (lot 426-27, expiration date, March 1986) was obtained from Cutter Medical Laboratory, Inc, Emeryville, CA. Manufacturing Division, Fair Lawn, NJ

MAXIMUM DOSE VOLUME APPLIED: 0.27 ml

DOSAGE PREPARATION (if unusual):
Five dosing suspensions (102.5, 129.0, 162.5, 204.5 and 258.0 mg/ml) were prepared by mixing an appropriate quantity of Guanidine Nitrate in 25 ml of MC-TW80 vehicle. The compound was ground to a fine powder with mortar and pestle and then slowly added to warm (50-60°C) MC-TW80. These
mixtures were vortexed, stirred, and sonicated to ensure good suspensions were achieved. Suspensions were evaluated for homogeneity and concentration. Samples for analysis were transferred to screwcapped tubes and stored below 0°C before analysis.

Doses:

Males and Females: 708 - 891 - 1121 - 1410 - 1780 mg/kg bw

No. of animals per sex per dose:

10 male and female animals, except:
Group 3: females totaled 14 - Group 4: females totaled 15 - Group 6: females totaled 7

Control animals:

other: yes, vehicle control group: 5 Males + 5 Females

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

Statistical analyses were performed on the study results. Selected lethal doses were derived by Litchfield-Wilcoxon probit analysis. The program, EASYGRAPH (Tektronix, Beaverton, OR) of the Data General Computer, Model MV8000, was used to draw the probit curve based on results from the Litchfield-Wilcoxon analyses.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

1 105 mg/kg bw

95% CL:

>= 957 - <= 1 276

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

1 028 mg/kg bw

95% CL:

>= 752 - <= 1 404

Mortality:

Fifty-one (91.1 %) of the deaths occurred within 4 hours after dosing. The remaining five deaths occurred within 24 hours after dosing. Two misdosed female mice were removed from the study and excluded from statistical analysis.
Thirty female mice died during the study. Of the 25 surviving female mice, 17 were normal within 24 hours and another 7 mice were normal within 48 hours. Twenty-six male mice died during the study. Of 24 surviving males, 14 were normal within 24 hours, and 8 other mice were normal within 48 hours. All vehicle control animals survived until study termination at 14 days.

Clinical signs:

other: The most frequently observed categories of clinical signs in animals administered Guanidine Nitrate were behavioral disturbances (51 of 105 animals dosed), hunched posture (30 of 105), and changes in reflex activity (26 of 105). Behavioral signs included

Gross pathology:

Vehicle control animals were normal (NLR = no lesion recorded) by gross examination. The mortalities in the groups which received compound appear to have been caused by the test compound except for one female identified as misdosed. Test compound-related lesions were found only
in the respiratory system. The lungs of 19 females and 10 males were congested. A single male in Group 6 (1780 mg/kg) had multiple petechiae in the glandular mucosa of the stomach which was considered an incidental finding.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 values for Guanidine Nitrate were determined to be 1105 mg/kg for male and 1025 mg/kg for female ICR mice.
Guanidine Nitrate is classified with Hazard Category 4 for acute oral toxicity (harmful if swallowed).

Executive summary:

The LD50 values for Guanidine Nitrate were determined to be 1105 mg/kg for male and 1025 mg/kg for female ICR mice.

Guanidine Nitrate is classified with Hazard Category 4 for acute oral toxicity (harmful if swallowed).

Reference 6

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

data not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

study was performed prior to guideline publication

Deviations:

yes

Remarks:

parameters assessed limited as compared to guideline

Principles of method if other than guideline:

The only parameters assessed were: mortality, macroscopic observations and autopsy.
Except for one table on mortality no other results were detailed in the report.
Only very brief summary report available.

GLP compliance:

no

Remarks:

study was performed prior to GLP legislation

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

derived strain from the colony of the test facility

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: colony of the testing facility: TNO (CENTRAL INSTITUTE FOR NUTRITION AND FOOD RESEARCH), Zeist, The Netherlands
- Weight at study initiation: Males 122 to 257 g - Females 116 to 162 g
- Fasting period before study: 16 hours
- Diet: ad libitum after treatment: stock diet
- Water: ad libitum after treatment: tap water

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

aqueous solution

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 20 % (w/v) of the test substance in a 0.5 % aqueous solution of carboxy methyl cellulose
- Amount of vehicle (if gavage): 4, 5, 6, 7, 8 ml/kg bw

MAXIMUM DOSE VOLUME APPLIED: 8 ml/kg bw i.e. 2.056 ml for a male animal weighing 257 g

Doses:

800 - 1000 - 1200 - 1400 - 1600 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Necropsy of survivors performed: yes
- Other examinations performed: gross clinical signs

Statistics:

The LD50 value was calculated according to the method of Litchfield and Wilcoxon (J. Pharmacol. exptl Therap. 96 (1949), 99-113.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

1 260

95% CL:

>= 1 086 - <= 1 461

Mortality:

800 mg/kg bw 0/10 (0 %)
1000 mg/ kg bw 0/5 M and 4/5 F (40 %)
1200 mg/ kg bw 1/5 M and 4/5 F (50 %)
1400 mg/ kg bw 2/5 M and 2/5 F (40 %)
1600 mg/ kg bw 4/5 M and 5/5 F (90 %)

Clinical signs:

other: During the first hours after treatment none of the animals showed any visible reactions to the treatment. Thereafter all animals became sluggish and looked sick. Several of them died during the first day. The survivors recovered rather quickly and no more

Gross pathology:

No abnormalities were seen in the surviving animals at autopsy.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 value of the Guanidine Nitrate as a mean value for males and females was calculated to be 1260 mg/kg body weight, with 1461 and 1086 as 95 % confidence limits. Guanidine Nitrate is classified with Hazard Category 4 for acute oral toxicity (harmful if swallowed).

Executive summary:

The LD50 value of the Guanidine Nitrate as a mean value for males and females was calculated to be 1260 mg/kg body weight, with 1461 and 1086 as 95 % confidence limits. Guanidine Nitrate is classified with Hazard Category 4 for acute oral toxicity (harmful if swallowed).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

729.3 mg/kg bw

Quality of whole database:

Quality ist very good, based on guideline studies

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

end of 1988 - early 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 180 to 200 g
- Fasting period before study: not mentioned
- Housing: single room, groups of 5 by sex, grid-floor stainless steel cages
- Diet (e.g. ad libitum): not mentioned
- Water (e.g. ad libitum):not mentioned
- Acclimation period: at least 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C, at several occasions 18 to 29 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

head only

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical aluminium exposure chamber
- Exposure chamber volume: 40 L
- Method of holding animals in test chamber: restrained in plastic tubes, nares only project into the chamber
- Source and rate of air: clean dry filtered air, diluent air flow 10.0 to 10.3 L/min
- Method of conditioning air: compression
- System of generating particulates/aerosols: Wright dust feed generator
- Method of particle size determination: Delcron CS5 Cascade Impactor with 5 separation stages (0.5, 1.0, 2.0, 4.0, 8.0 μm)
- Temperature, humidity in air chamber: 18 to 20 °C, 37 to 55 % humidity

TEST ATMOSPHERE
- Brief description of analytical method used: For the determination of the nominal concentration, the total weight of test article used and the volume Q of diluent air used to generate the test atmosphere were recorded and the nominal concentration of the test article in the exposure chamber was calculated as follows:
Nominal concentration (mg/L) = weight of test article used (mg) / flowrate (L/min) x duration (min)
- Samples taken from breathing zone: not specified

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: particle size analysis was performed
- MMAD (Mass median aerodynamic diameter): 5.52 μm

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

The study was performed as a limit test.
mean measured concentration: 0.853 mg/L
nominal concentration: 17.273 mg/L

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: hourly during exposure and for the remainder of the exposure day then daily for 14 days
- Frtequency of weighing: day of dosing, days 8 and 15 of the study
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, lung weight

Statistics:

means and standard deviations calculated where appropriate

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 0.853 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 17.273 mg/L air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were no deaths.

Clinical signs:

other: Slight clinical signs of staining and wetting of the fur in the test group disappeared by day 4.

Body weight:

There was no treatment-related effect on body weight.

Gross pathology:

There were no treatment-related changes in lung weight or in macroscopic appearance at necropsy.

Interpretation of results:

GHS criteria not met

Conclusions:

The LC 50 of Guanidine Nitrate was determined to be > 0.853 mg/L air (analytical) in this study. This concentration was the maximum possible under conditions of this study, generated from a nominal concentration of 17.273 mg/L. No animal died and no treatment related clinical signs were observed.

Executive summary:

In an acute inhalation toxicity study performed similar to OECD Guideline 403 5 male and 5 female young adult Crl:CD(SD)BR rats were exposed by inhalation route to micronized Guanidine Nitrate (99.3 % a.i.) for 4 hours to head only at a chamber concentration of 0.853 mg/L. The corresponding nominal concentration was 17.273 mg/L and the mass median aerodynamic diameter of the particles in the atmosphere was 5.52 µm. The nominal concentration was the maximum practical for the generator used and the resulting analytical concentration the maximum possible under conditions of this study. Animals then were observed for 14 days.

LC₅₀      Combined =  > 0.853 mg/L air (analytical)

             Combined =  > 17.273 mg/L air (nominal)

No animals died and there were no treatment related clinical signs, necropsy findings( including lung weights) or changes in body weight, exceptof staining and wetting of the fur, which disappeared by day 4.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Supporting information for read-across, structural analogue approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: 140 - 200 g
- Fasting period before study: no data
- Housing: groups of 5 animals by sex in grid-floor stainless steel cages
- Diet (e.g. ad libitum): ad libitum; SQC Rat and Mouse Maintenance Diet No. 1 Expanded, Special Diets Services Ltd. Witham
- Water (e.g. ad libitum): ad libitum; mains water
- Acclimation period: 25 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19 - 25 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

head only

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical anodised aluminium exposure chamber
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: head-only
- Source and rate of air: The compressed air supply used was from a clean dry filtered source.
- Method of conditioning air: -
- System of generating particulates/aerosols: DeVilbiss model 646 liquid nebulizer generator, dynamic (continuous flow)
- Method of particle size determination: The particle size was determined using a Delron C55 Cascade Impactor, with 5 separation
stages corresponding to maximum mass median aerodynamic diameters of 0.5, 1.0. 2.0. 4.0 and 8 µm. The samples were obtained
hourly, over periods of up to 8 minutes, -during the exposure period.
- Treatment of exhaust air: The atmospheres were filtered, exhausted to the outside of the building and vented.
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity inside the exposure
chamber were measured continuously and recorded at hourly interva1s throughout th'e 4-hour exposure peri od, using a
digital thermometer with remote probe located in the chamber and a paper hygrometer located in the exhaust duct of the chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the test article was determined gravimetrically. The atmosphere
was sampled by drawing a known volume through an open face glass fibre filter positioned at a site representative of that
occupied by the external nares of the experimental animals.
- Samples taken from breathing zone: yes



TEST ATMOSPHERE (if not tabulated)
- Particle size distribution:
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

0.513, 2.118, 4.585 and 6.518 mg/L

No. of animals per sex per dose:

5 aminals per sex per group

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: twice daily for dead or moribund. The body weight of each animal was recorded immediately before and
after exposure, on days 8 and 15 of the study and at necropsy.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other:
Clinical signs were observed at hourly intervals during the exposure period, then for the remainder of the working day, and once daily thereafter for
14 days.
Necropsy: A full internal and external examination was made under the general supervision of a pathologist. The nasal passages were examined and
an assessment made of any irritation of the respiratory tract.
Organ weights: The lungs, bronchi and trachea were dissected free from fat and other contiguous tissue and weighed together.
Histology: Samples of all gross lesions were fixed in 10% neutral buffered formalin and retained without further processing.

Statistics:

The median lethal concentrations (LC50) together·with 95% fiducial limits were calculated separately for males and females, and for
sexes combined, from the recorded mortality rate using a probit analysis method (Finney, D.J. (971)
Probit Analysis, 3rd ed., Cambridge University Press).
,I
,I

Key result

Sex:

female

Dose descriptor:

LC50

Effect level:

3.181 mg/L air (analytical)

Based on:

test mat.

95% CL:

>= 0.567 - <= 50.411

Exp. duration:

4 h

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

7.655 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

5.319 mg/L air (analytical)

Based on:

test mat.

95% CL:

>= 2.981 - <= 34.296

Exp. duration:

4 h

Mortality:

Qveral-l there was a positive correlation between mortality and chamber concentration. As the minimum level at which death
occurred was 4.585 mg/l for males and 2.118 mg/l for females, the possibility of a sex difference cannot be eliminated.
One male in group 5 died during exposure. Except for one death on day four all deaths occurred on days one or two of the study.
Seven animals were killed for humane reasons on day one. Five of these decedents were in moribund condition.

Clinical signs:

other: Marked signs of toxicity were observed at 4.585 and 6.518 mg/L immediately after exposure. The signs included ataxia, occasionally prostration and irregular respiration. and convulsion and signs of ejaculation at 4.585 mg/l. During and/or following expos

Body weight:

Body weight losses occurred as a result of the r~straint procedures in test and control groups. The losses tended to be greater in
treated groups and persisted in a few individuals until day 8 of the study. However, overall there was no marked effect on body
weight.

Gross pathology:

Mean absolute and relative lung weights at termination for treated groups tended to be slightly higher than the corresponding control va1ues. but all values fell withi n the normal range and the numerical differences were too small to conclude there was a treatment-related effect.
Occasional lung weights for decedents were near or ablove the upper limits of the normal range.
This was probably due to agonal congestive changes.
Animals surviving to termination showed only incidental changes. Some decedents were unremarkable but others had discoloured and
inflated lungs suggestive of acute effects on the cardiopulmonary system. Four of the flve group 3 females had sore forefeet
possible due to self-inflicted trauma.

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

CLP, EU GHS (Regulation (EC) No. 1272/2008)

Conclusions:

Based on the LC50 female of 3.181 mg/L air the test substance has to be classified according to CLP, EU GHS (Regulation (EC) No. 1272/2008) in Toxicity Category 4.

Executive summary:

In an acute inhalation toxicity study performed according to OECD Guideline 403 5 young Sprague-Dawley rats per sex per group were exposed at single chamber concentrations of 0.513, 2.118, 4.585 and 6.518 mg/L of Guanidine hydrochloride (98.5 % a.i.) by inhalation (head-only) over a period of 4 hours. Corresponding nominal concentrations were in the range 2.124 to 15.280 mg/L. The mass median aerodynamic diameters of the particles in the atmospheres fell in the range 1.88 to 5.62 µm. The aerosol for animal exposures was prepared from an aqueous solution of the test article. A similar group of 5 males and 5 females was exposed to filtered air as a control. Animals then were observed for 14 days.

The acute inhalation median lethal concentrations, calculated by a probit method, were:

LC50 combined: 5.319 mg/L

LC50 Males: 7.655 mg/L

LC50 Females: 3.181 mg/L

Deaths occurred in males at a level of 4.585 mg/l and above, and in females at a level of 2.118 mg/l and above. Overall there was a dose-related relationship between mortality and chamber concentration. Nearly all deaths occurred on days one or two of the study. Marked clinical signs were first observed on the day of exposure. The signs included ataxia and occasionally prostration, irregular respiration, convulsion and signs of ejaculation. A few individuals showed body weight loss that persisted one week after treatment, but overall there was no marked treatment related effect on body weight.

There was no evidence of a treatment-related, effect on lung weight in survivors. Occasional increases in lung weights in decedents were probably due to agonal congestive changes. Animals surviving to termination were unremarkable macroscopically. A few decedents had discolored and inflated lungs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

3 181 mg/m³ air

Quality of whole database:

Quality ist very good, based on guideline study

Acute toxicity: via dermal route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-06-21 to 1984-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Health effects test guidelines, August 1982, EPA 560/6-82-001

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, Watsonville, CA
- Weight at study initiation: 2.7 to 3.7 kg
- Housing: individually in steel wire bottem cages in racks equipped with automatically flushing dumptanks
- Diet: approximately 150 g of Certified Purina Rabbit Chow Diet 5322 (Ralston Purina Company, St. Louis, MO)
- Water ad libitum: provided by continous drip from a central line
- Acclimation period: 25 days
- Earmite prevention during acclimation: one application of Canex mineral oil (Pitman-Moore, Inc., Washington Crossing, NJ)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22
- Humidity (%): 50 to 66 with ocasional spike sduring room cleaning
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1984-06-21 To: 1984-07-31

Type of coverage:

semiocclusive

Vehicle:

physiological saline

Remarks:

(0,9 % sodium chloride for injection, USB, Travenol Laboratories, Inc., Deerfield, IL, Lot No. 7C50X0, Expiration date . October 1985)

Details on dermal exposure:

TEST SITE
- Area of exposure: 300 cm²
- Type of wrap if used: Vetrap® bandaging tape (Animal Care Products, 3M Corp., St. Paul, MN)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): wiping with a piece of gauze moistened with 0.9 % saline
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 6.19 to 7.49 g in 10 ml of vehicle
- Constant volume used: yes
- For solids, paste formed: yes

VEHICLE
- Amount applied (volume with unit): 10 ml
- Concentration (if solution): 0.9 % sodium chloride (USB, Travenol Laboratories, Inc., Deerfield, IL)
- Lot/batch no. (if required): Lot No. 7C50X0, Expiration date . October 1985

Duration of exposure:

24 hours

Doses:

limit dose at 2000 mg / kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: daily
- Frequency of weighing: weeky
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs (systemic and dermal), body weight, histopathology

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occured during the study.

Clinical signs:

other: Systemic: None of the clinical systemic signs were interpreted as signs of toxicity attributable to the test compound. Dermal: Signs associated with dermal toxicity included erythema, edema, and necrosis, dermal irritation was scored at 1/2, 24, 48. and

Gross pathology:

No deaths occured during the study. Guanidine Nitrate did not produce pathological changes observable at necropsy. Sections of treated and control skin were examined microscopically and no treatment-related lesions were found.

Other findings:

Guanidine Nitrate was a dermal irritant under conditions of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

On the basis of the results obtained after a single dermal administration, the dermal LD50 of Guanidine Nitrate was determined to be > 2000 mg/kg bw. No animal died. No clinical signs or gross pathological findings were observed which would indicate systemic toxicity. Body weight was not influenced by the treatment.

Executive summary:

In an acute dermal toxicity study according to the Health effects test guideline, August 1982, EPA560/6-82-001, 5 male and 5 femaleyoung adult New Zealand White rabbits were dermally exposed to guanidine nitrate (99.99 % a.i) in 0.9 % sodium chloride solution for 24 hours to approx. 300 cm² of body surface area at a dose of 2000 mg/kg bw under an semiocclusive dressing.  Animals then were observed for14days.

 

Dermal LD₅₀      Combined =   > 2000 mg/kg bw

 No mortality occurred in this limit test.There were no treatment related systemic clinical signs, necropsy findings or changes in body weight. Dermal irritation was observed including very slight to moderate erythema and very slight to slight oedema. Symptoms were reversible within the observation period. Necrosis was observed in one male which persist as a scab for the entire 14 days of the study. At terminal necropsysections of treated and control skin were examined microscopically and no treatment-related lesions were found.

Reference 2

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984-05-18 to 1984-08-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Supporting information for read-across, structural analogue approach.

Qualifier:

according to guideline

Guideline:

other: EPA TS-792 Acute exposure, dermal toxicity. Health effects test guidelines, EPA, August 1982; EPA 560/6-82-001

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, 5265 Starr Way, Watsonville, CA 95076
- Age at study initiation: young adults
- Weight at study initiation: 2.8 to 3.7 kg
- Housing: individually
- Diet (e.g. ad libitum): 150 g of Certified Purina Chow Diet 5322
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 25 d (quarantine, acclimation)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 24°C
- Humidity (%): 46 to 74%, except for spikes up to 94% during cleaning
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

physiological saline

Details on dermal exposure:

TEST SITE
- Area of exposure: 240 cm²
the substance was applied on gauze dressing, taped with hypoallergenic tape (Durapore) and wrapped with bandage (Vetrap)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: after 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- For solids, paste formed: yes, 0.9% saline

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: clinical signs and mortality daily; dermal reactions 0.5, 24, 48, 74 h after removal of the patches; body weights were recorded 7 times during test period
- Necropsy of survivors performed: yes
- Other examinations performed: skin from exposed area was examined microscopically

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

no mortality occurred

Clinical signs:

other: systemic: - no substance-related clinical signs local (dermal): - erythema was observed in all rabbits 30 min after patch removal; 24 h after patch removal the erythema had disappeared in five animals - slight erythema, necrosis and ulceration was observ

Gross pathology:

- epidermal ulceration covered by a fibrinocellular exudate in one male with necrosis and eschar formation
- no other gross findings

Interpretation of results:

GHS criteria not met

Conclusions:

The dermal LD50 of Guanidine hydrochloride in rabbits was > 2000 mg/kg bw.

Executive summary:

In an acute dermal toxicity study similar to OECD guideline 402 groups of 5 young adult New Zealand White rabbits/sex were dermally exposed to Guanidine hydrochloride (98% pure) moistened with 0.9% saline for 24 h to 240 cm² body surface area at a limit dose of  2000  mg/kg bw using occlusive dressing.  Animals then were observed for 14 days.

No mortality occurred. No substance-related systemic effects were observed. Exposure to the test substance caused dermal irritation and eschar formation.

Dermal LD50 > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Quality ist very good, based on guideline study

Additional information

Acute toxicity: oral

In the key study for acute oral toxicity, performed according to the Health effects test guidelines, August 1982, EPA 560/6-82-001, groups of Sprague Dawley rats (10/sex) were given a single oral dose of Guanidine Nitrate (99.99 % a.i.) in methylcellulose, Tween® 80, sterile water at doses of 311, 826, 1000, 1210 and 1470 mg/kg bw (males) and 610, 718, 847, 1000, 1180 and 1390 mg/kg bw (females) and observed for 14 days.

Oral LD50 Males = 989.6 ± 68.7 mg/kg bw (95% C.I. > 793 - 1138)

Females = 729.8 ± 34.3 mg/kg bw (95% C.I. > 641 - 799)

65 animals died during the study, most of them during the first 8 hours after dosing. Weight gains of surviving animals were not significantly affected by dosing. The primary category of clinical signs was behavioral (e.g., inactive, irritable, disoriented, hyperactive, ataxic) which was observed in 63 of 99 animals dosed with Guanidine Nitrate. Other categories of frequently observed clinical signs were gastrointestinal (e.g., material in mouth, perlanal staining, increased salivation, diarrhea) which was observed in 37 of 99 animals, and respiratory (e.g. reddish nasal discharge, increased rate and/or decreased depth of respiration) which was observed in 26 of 99 animals. These findings suggest that guanidine nitrate exhibits a primary effect on the central nervous / neuromuscular system. The presence of multiple red foci in the thymuses of the females was dose related and the only gross lesion which was attributable to the test compound.

In two supporting studies for acute oral toxicity, the LD50 values for Guanidine Nitrate were determined to be 1105 mg/kg for male and 1025 mg/kg for female ICR mice.

Furthermore, the LD50 value of the Guanidine Nitrate as a mean value for male and female rats was calculated to be 1260 mg/kg body weight, with 1461 and 1086 as 95 % confidence limits.

Acute toxicity: inhalation

In an acute inhalation toxicity study performed similar to OECD Guideline 403 5 male and 5 female young adultCrl:CD (SD)BR rats were exposed by inhalation route to micronized Guanidine Nitrate (99.3 % a.i.) for 4 hours to head only at a chamber concentration of 0.853 mg/L. The corresponding nominal concentration was 17.273 mg/L and the mass median aerodynamic diameter of the particles in the atmosphere was 5.52 µm. The nominal concentration was the maximum practical for the generator used and the resulting analytical concentration the maximum possible under conditions of this study. Animals then were observed for 14 days.

LC50 Combined = > 0.853 mg/L air (analytical)

Combined = > 17.273 mg/L air (nominal)

No animals died and there were no treatment related clinical signs, necropsy findings( including lung weights) or changes in body weight, except of staining and wetting of the fur, which disappeared by day 4.

Additional acute inhalation toxicity data are available for the read-across substance guanidine hydrochloride.

In an acute inhalation toxicity study performed according to OECD Guideline 403 5 young Sprague-Dawley rats per sex per group were exposed at single chamber concentrations of 0.513, 2.118, 4.585 and 6.518 mg/L of Guanidine hydrochloride (98.5 % a.i.) by inhalation (head-only) over a period of 4 hours. Corresponding nominal concentrations were in the range 2.124 to 15.280 mg/L. The mass median aerodynamic diameters of the particles in the atmospheres fell in the range 1.88 to 5.62 µm. The aerosol for animal exposures was prepared from an aqueous solution of the test article. A similar group of 5 males and 5 females was exposed to filtered air as a control. Animals then were observed for 14 days. The acute inhalation median lethal concentrations, calculated by a probit method, were:

LC50 combined: 5.319 mg/L air (analytical)

LC50 males: 7.655 mg/L air (analytical)

LC50 females: 3.181 mg/L air (analytical)

Deaths occurred in males at a level of 4.585 mg/L and above, and in females at a level of 2.118 mg/L and above. Overall there was a dose-related relationship between mortality and chamber concentration. Nearly all deaths occurred on days one or two of the study. Marked clinical signs were first observed on the day of exposure. The signs included ataxia and occasionally prostration, irregular respiration, convulsion and signs of ejaculation. A few individuals showed body weight loss that persisted one week after treatment, but overall there was no marked treatment related effect on body weight. There was no evidence of a treatment-related, effect on lung weight in survivors. Occasional increases in lung weights in decedents were probably due to agonal congestive changes. Animals surviving to termination were unremarkable macroscopically. A few decedents had discolored and inflated lungs.

Acute toxicity: dermal

In the key study for acute dermal toxicity, performed according to the Health effects test guideline, August 1982, EPA560/6-82-001, 5 male and 5 female young adult New Zealand White rabbits were dermally exposed to guanidine nitrate (99.99 % a.i) in 0.9 % sodium chloride solution for 24 hours to approx. 300 cm² of body surface area at a dose of 2000 mg/kg bw under an semiocclusive dressing. Animals then were observed for14 days.

Dermal LD50 Combined = > 2000 mg/kg bw

No mortality occurred in this limit test. There were no treatment related systemic clinical signs, necropsy findings or changes in body weight. Dermal irritation was observed including very slight to moderate erythema and very slight to slight oedema. Symptoms were reversible within the observation period. Necrosis was observed in one male which persist as a scab for the entire 14 days of the study. At terminal necropsy sections of treated and control skin were examined microscopically and no treatment-related lesions were found.

Additional supporting data from the read-across substance guanidine chloride were included to demonstrate the similar toxicological profile of both substances. Data were used to substantiate the justification for read-across.

Justification for read-across:

Guanidine hydrochloride and guanidine nitrate dissociate in biological fluids to yield the guanidine ion and the respective anion. Therefore it is reasonable to discuss the systemic intrinsic properties of the ions separately. The chloride ion is a naturally occurring essential ion in human beings with well-known metabolism and mechanisms of action as described in standard textbooks on pharmacology and physiology. Systemic effects of guanidine hydrochloride are expected to be based on the guanidine ion. The physiological processing of the guanidine ion is expected to be independent of the individual source. Therefore read-across from guanidine hydrochloride for acute systemic effects of guanidine dissociated from guanidine nitrate after inhalation is considered valid.

This strategy is supported by a quite similar toxicological profile of both substances, as shown in acute toxicity, irritation, sensitization and genotoxic studies.

A more detailed justification is attached and outlined in CSR chapter 1.1.2 as well.

Justification for classification or non-classification

According to CLP, EU GHS (Regulation (EC) No 1272/2008), Guanidine Nitrate is classified with Hazard Category 4 for acute oral toxicity (harmful if swallowed) since the results obtained in all available studies are conclusive and L50 values are in the range from 300 to 2000 mg/kg body weight.

The findings from acute oral toxicity studies suggest that the guanidine ion exhibits a primary effect on the central nervous / neuromuscular system. However, effects were observed in the range of classification Hazard Category 4, therefore on the basis of available data no additional classification for STOT-SE is considered necessary.

A sub-chronic repeated dose toxicity study is proposed, which will provide additional more detailed information for evaluation.

Guanidine Nitrate is classified for acute inhalation toxicity with Hazard Category 4. Data from a study conducted with the read-across substance guanidine hydrochloride with a LC50 value for females of 3.181 mg/L air, indicating the need for classification. However, in the study with guanidine nitrate at the maximum achievable effective dose of 0.853 mg/L air (analytical; corresponding nominal concentration was 17.273 mg/L) no deaths occurred.

Guanidine Nitrate is not classified for acute dermal toxicity since the LD50 value is > 2000 mg/kg body weight.

Guanidine Nitrate is classified with R20/22 according to Directive 67/548 EEC.