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Description of key information

The substance was found to be not irritating in an in vitro skin irritation test, using a human skin model (EPISKIN Standard Model). In a screening assay using the bovine corneal opacity and permeability test (bcop test), the test substance was found not to be  a severe irritant or have corrosive properties. In an eye irritation study with rabbits, performed according to OECD/EC test guidelines, limited irritation was observed, not sufficient for classification. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test), adopted 24 August 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted 22 July 2010
Deviations:
no
GLP compliance:
yes
Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C):35.9 – 37.6
- Humidity (%): 85-96
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 10.8 to 13.6 mg (evenly distributed over the tissue)

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Standard ModelTM(EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-39).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days , which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation parameter:
other: tissue viability
Basis:
other: percentage of control
Time point:
other: 15 minutes
Score:
112
Remarks on result:
other: Negative control = 100%; Positive control =9%

Guanidine phosphate (1:1) was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that Guanidine phosphate (1:1) did not interact with MTT.

 

Mean tissue viability for the test substance was > 50%, therefore the test substance is considered not to be irritant to the skin.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on an in vitro skin irritation test performed according to OECD 439 guideline and GLP principles, it is concluded that guanidinium phosphate (1:1) is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of guanidinium phosphate (1:1) on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin ( 10.8 to 13.6 mg). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 9% whereas the test substance showed cell viability of 112%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the guanidinium phosphate (1:1) is not irritating in the in vitro skin irritation test.

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:
14JAN2013 to 24JAN2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 2011, including the most recent partial revisions.
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: Animals used withihn the study were 13-15 weeks old
- Weight at study initiation: Body weights were 2.471-2.916 kg
- Housing: Individually housed in cages with perforated floors
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy). Hay and wooden sticks were available during the study period
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14JAN2013 to 24JAN2013
Vehicle:
water
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit):
67.3 mg (range 67.1 – 67.5 mg); a volume of approximately 0.1 mL

Duration of treatment / exposure:
Single instillation on Day 1.
Observation period (in vivo):
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation of the test substance .
Number of animals or in vitro replicates:
3 males
Details on study design:
STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner one week later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Animals were treated by instillation of the test substance in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20%.

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS (beschrijf beperkt)
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to the scoring system as described in OECD guideline 405.
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
mean
Remarks:
of three animals
Time point:
other: 24, 48 and 72 hrs.
Score:
0
Max. score:
4
Remarks on result:
other: No effects on the cornea were observed.
Irritation parameter:
iris score
Basis:
mean
Remarks:
of three animals
Time point:
other: 24, 48 and 72 hrs.
Score:
0
Max. score:
2
Remarks on result:
other: No effects on the iris were observed.
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
mean
Remarks:
of three animals
Time point:
other: 24, 48 and 72 hrs.
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of three animals
Time point:
other: 24, 48 and 72 hrs.
Score:
0.11
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Remarks:
(discharge)
Basis:
mean
Remarks:
of three animals
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
3
Remarks on result:
other: No discharge was observed.
Irritant / corrosive response data:
Instillation of approximately 67 mg of Guanidine phosphate (1:1) (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 48 hours in one animal and within 72 hours in the other two animals.
No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.
There was no evidence of ocular corrosion.
Other effects:
No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an eye irritation study with rabbits, performed according to OECD/EC test guidelines, limited irritation was observed.
Executive summary:

An eye irritation study was performed with rabbits, performed according to OECD/EC test guidelines. Approximately 67.3mg Guanidinium phosphate (1:1) in a volume of approximately 0.1 mL was instilled in one eye of each of the three rabbits. This resulted in irritation of the conjunctivae, consisting of redness, chemosis and discharge. The irritation had completely resolved within 48 hours in one animal and within 72 hours in the other two animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. There was no evidence of ocular corrosion.

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. Based on these data, guanidinium phosphate is considered not to be irritating to the eye and is not classified according to Regulation (EC) 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of guanidinium phosphate (1:1) on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2cultured skin ( 10.8 to 13.6 mg). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 9% whereas the test substance showed cell viability of 112%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the guanidinium phosphate (1:1) is not irritating in the in vitro skin irritation test.

The eye irritancy potential of guanidine phosphate (1:1) was assessed using the Bovine Corneal Opacity and Permeability test (BCOP test). The negative and positive control responses of the opacity and permeability values were within the range of the laboratory historical range indicating that the test system functioned properly (positive control: 103; negative control: 0).

Guanidine phosphate (1:1) did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 4.0 after 240 minutes of treatment. Based on these data, it can be concluded that guanidine phosphate (1:1) is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report. An eye irritation study was performed with rabbits, performed according to OECD/EC test guidelines. Approximately 67.3mg Guanidinium phosphate (1:1) in a volume of approximately 0.1 mL was instilled in one eye of each of the three rabbits. This resulted in irritation of the conjunctivae, consisting of redness, chemosis and discharge. The irritation had completely resolved within 48 hours in one animal and within 72 hours in the other two animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. There was no evidence of ocular corrosion. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Justification for selection of skin irritation / corrosion endpoint:
Only one study available; This study has been performed according to OECD and/or EC guidelines and according to GLP principles (reliability 1).

Justification for selection of eye irritation endpoint:
This study has been performed according to OECD and/or EC guidelines and according to GLP principles (reliability 1). Also a BCOP assay was performed to screen the irritating potential of the test substance with reliability 1, which is used as supporting data.

Justification for classification or non-classification

Based on the available data, guanidinium phosphate is considered not to be irritating to the skin or to the eye and is not classified according to Regulation (EC) 1272/2008.