1-nitroguanidine

Endpoint summary

Description of key information

Two U.S. EPA Guideline studies are available covering primary dermal irritation potential and eye irritation potential

in rabbits. Nitroguanidin is shown not to be irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1984 - April 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA 560/6-82-001

Deviations:

yes

Remarks:

(The rabbits were weighed on 3 October instead of 2 October. This had no effect on the results of the study. )

GLP compliance:

yes

Remarks:

GLP Study Report

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, Watsonville, CA; U.S.A.
- Age at study initiation: young adults
- Weight at study initiation: 3.2-3.6 kilograms
- Housing: individually in stainless steel, screenbottomed, battery-type cages with automatically flushing dump tanks
- Diet: 150 g/d Certified Purina Chow Diet 5322 (Ralsotn Purina Company, St.Loius, MO)
- Water (e.g. ad libitum): tap water
- Acclimation period: for 5 days prior to testing following a 14 day quarantine, during this period they were observed daily for signs of illness

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-20.0 °C
- Humidity (%): 40-70%
- Air changes (per h): no data
- Photoperiod (hrs dark/hrs light): 12/12


IN-LIFE DATES: From: 06/09/1984 To: 09/10/1984

Type of coverage:

occlusive

Preparation of test site:

other: close-clipped

Vehicle:

physiological saline

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g


VEHICLE
- Amount applied: enough to moisten 0.5 g of Nitroguanidine and form a thick paste
- Concentration of saline solution: 0.9%
- Lot/batch no.: 8C865AY

Duration of treatment / exposure:

4 hours

Observation period:

1 hour and 24, 48 and 72 h after treatment

Number of animals:

eight (four male and four female rabbits)

Details on study design:

TEST SITE
- Preparation of test site: backs were pre-clipped 4 days before dosing and then close-clipped 24 hours before actual dosing
- Area of exposure: back; the clipped area was devided into 4 qaudrants designated I, II, III, and IV
- Site I: vehicle control site
- Sites II and III: test compound sites
- Site IV: sham patch site
- % coverage:
- Type of wrap if used: gauze patch which was taped (semi-impervious hypo-allergenic surgical tape) to the appropriate site; Vet Wrap was then wrapped securely around the animal


REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 4 h
- test substance was removed, the skin was wiped if the material adhered


SCORING SYSTEM: see table 1

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 30-60 min

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 30-60 min after treatment

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

- no irritation/corrosion occured (see table 2)

Other effects:

Rabbit number 570 had multiple small (1 mm) pustules ventral to the patch area at 48 and 72 h after dosing. These lesions were positive for Staphylococcus aureus on bacteriological culture. The test area was not involved.

Table 2: Summary of Primary Irritation Test Data

	30-60 min			24 h			48 h			76 h
Animal No.	Test	Sham	Vehicle	Test	Sham	Vehicle	Test	Sham	Vehicle	Test	Sham	Vehicle
570	0	0	0	0	0	0	0	0	0	0	0	0
571	0	0	0	0	0	0	0	0	0	0	0	0
572	0	0	0	0	0	0	0	0	0	0	0	0
573	0	0	0	0	0	0	0	0	0	0	0	0
574	0	0	0	0	0	0	0	0	0	0	0	0
575	0	0	0	0	0	0	0	0	0	0	0	0
577	0	0	0	0	0	0	0	0	0	0	0	0
578	0	0	0	0	0	0	0	0	0	0	0	0
Mean*	0	0	0	0	0	0	0	0	0	0	0	0
Net Mean Score t	0	0	0	0	0	0	0	0	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

Nitroguanidine is not irritating under the conditions of this assay.

Executive summary:

This study has been performed in compliance with Good Laboratory Practice (GLP). The study has been conducted according to US EPA Guideline 560/6-82-001, adopted August 1982.

A gauze patch with 0.5 g of test material (Nitroguanidine), moistened with 0.9% (w/v) physiol. saline, was placed on the clipped skin of four male and female New Zealand White rabbits and closely covered withgauze patch which was taped (semi-impervious hypo-allergenic surgical tape) to the appropriate site. Then Vet Wrap was wrapped securely around the animals. Total exposure duration was four hours. The control site was treated with physiol. saline only and covered analogically.

The animals were checked daily for mortality and systemic symptoms. Body weights were recorded four days prior to testing, at study initiation, and five and eight days after treatment. Skin reactions were evaluated 1, 24, 48, and 72 h after removing the patches according to the scoring system in table 1.

The test-compound was non-irritating. Neither erythema, edema, nor any other recognisable skin reactions were detected at 1, 24, 48 or 72 h after dosing or during the remaining period before terminal sacrifice 14 days after dosing.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1984 - January 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA 560/6-82-001

Deviations:

no

Remarks:

slit lamp exam. was added; colour photographic docum. was not performed; 8-hour period with altered temperatur and humidity range; diet was fed at 150 g/d and not ad libitum > none of these dev. had a negative impact on study performance and validity

GLP compliance:

yes

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Elkhorn Rabbitry, 5265 Starr Way, Watsonville, CA, U.S.A.)
- Age at study initiation: young adults
- Sex: males
- Weight at study initiation: 2.4-3.5 kg
- Housing: individually in stainless steel, screen-bottomed, battery-type cages with automatically flushing dump tanks
- Diet: approx. 150 g/day of Certified Purina Chow Diet 5322 (Ralston Purina Company)
- Water: continous drip from a central line
- Pretest conditioning: 1. quarantine from 6 Sept- 19 Sept 1984
2. animal eyes were examined 24 hours before dosing using slit lamp, fluorescein dye and ultraviolet light
- Acclimation period: at least five days after quarantine


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14.4-17.8; exception: 8-hour period on 22 Sep 84 when the temperature fluctuated between 12.8 and 26.7 °C
- Humidity (%): 40-70%
- Photoperiod (hrs dark/hrs light): 12/12


IN-LIFE DATES:
Group1 > From: 25 Sep 1984 To: 28 Sep 1984
Group2 > From: 2 Oct 1984 To: 5 Oct 1984

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.025 g (=0.1 ml)
- Administration: the lower lid was pulled away from the conjunctival cul-de-sac to form a cup into which the compound was dropped. Upper and lower lids were then held gently together for one second to prevent loss of material.

Duration of treatment / exposure:

single application

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three animals per dose group (two dose groups).
Group one was dosed on 25 Sep 84 and group 2 was dosed on 2 Oct 84.

Details on study design:

TEST PROCEDURE:
- prior to study initiation animals were examined for any pre-existing abnormalities.
- For each animal, the eye with the nearest normal appearance was designated for treatment; the contralateral eye served as control.
- On the next day, 0.025 g Nitroguanidine was placed in the treated eye of each rabbit in this group (group 1: 25 Sep 1984; group 2: 2 Oct 1984)


SCORING SYSTEM: Draize-type ocular grading


TOOL USED TO ASSESS SCORE:
- slit lamp (24, 48, and 72-hour-observations); additionally any areas appearing grossly abnormal were examined under high magnification
- fluorescein: introduced onto the eye which was then observed under unltraviolet light. Any corneal areas reacting with the dye (a sign of discontinuity of the corneal epithelium) were described with respect to area and intensity of fluorescence
- Examination and grading of ocular reactions were performed in this fashion at 1, 4, 24, 48, and 72 h after dosing.
- Fluorescein staining was omitted from the 1 and 4 hour-observations, as was slit lamp examination.
- Due to an almost lack of reaction during the 72 hours after dosing, the study was terminated, according to protocol, after this observation. Therfore no scoring or observations were performed at 7, 14, or 21 days.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 1 and 4 h after dosing

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 1 and 4 h after dosing

Score:

0

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Time point:

other: 1 and 4 h after dosing

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 1 day

Remarks on result:

other: slight conjunctival injection

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

conjunctival swelling

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

conjunctival swelling

Basis:

mean

Time point:

other: 1 and 4 h after dosing

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Control Eyes:

- At no time during the study did no contralateral, untreated eyes exhibit any changes from the pre-dosing examination.

- Fine diffuse stippling was present before and after dosing in the control eye of one rabbit.

- All other untreated controls were normal throughout the entire study.

Nitroguanidine produced no positive reactions. Slight conjunctival redness, indicating mild inflammation, was the only response observed. This reaction, although scorable, did not achieve sufficient severity to warrant consideration as a “positive response”. A likely explanation for this slight redness was the observed presence in the lower conjunctival cul-de-sac (the site of gradable redness) of small amounts of the test material. Most of the eyes with gradable redness exhibited undissolved nitroguanidine. Therefore physical irritation, not pharmacological activity, may have been responsible for the slight response observed. Since human exposure would be closely followed with thorough washing, even this slight response may not occur.

Interpretation of results:

GHS criteria not met

Conclusions:

In the present study, nitroguanidine produced no response indicative of a potential to cause occular irritation upon direct contact with the eye.

Executive summary:

In an acute eye irritation study using a modified Draize method (US EPA 560/6-82-001), 0.1 ml (corresponding to 0.025 g) of Nitroguanidine was instilled into the conjunctival sac of the eye of six male albino rabbits (New Zealand White strain). Eyes were not washed afterwards. Animals then were observed for 3 days. Irritation was scored by the method of US EPA 560/6-82-001.

Under the experimental conditions employed, Nitroguanidine produced no positive reactions. Slight conjunctival redness, indicating mild inflammation, was the only response observed. This reaction, although scorable, did not achieve sufficient severity to warrant consideration as a “positive response”. A likely explanation for this slight redness was the observed presence in the lower conjunctival cul-de-sac (the site of gradable redness) of small amounts of the test material. Most of the eyes with gradable redness exhibited undissolved nitro guanidine. Therefore physical irritation, not pharmacological activity, may have been responsible for the slight response observed. Since human exposure would be closely followed with thorough washing, even this slight response may not occur.

In the present study, nitroguanidine produced no response indicative of a potential to cause occular irritation upon direct contact with the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Nitroguanidine is tested non-irritating to the skin. No erythema, edema, or any other recognisable skin reactions have been detected at 1, 24, 48, or 72 hours after dosing 0.5 g NiGu(99 %) or during the remaining period before terminal sacrifice 14 days after dosing.

Nitroguanidine is tested non-irritating to the eye. Slight conjunctival redness, indicating mild inflammation, was the only response observed after instillation of 0.1 ml (25 mg) NiGu (99 %) into the conjunctival sac of six male New Zealand White rabbits during an observation period of 3 days.

Justification for selection of skin irritation / corrosion endpoint:
Reliable guideline study

Justification for selection of eye irritation endpoint:
Reliable guideline study

Justification for classification or non-classification

Nitroguanidin is not irritating/corrosive to the skin or irritating to the eyes based on the results of appropriate guideline studies.