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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Principles of method if other than guideline:
The study was conducted according to the method described by Hoover DM et al. (Comparison of the in vitro and in vivo models to assess venous irritation of parental antibiotics. Fundam. Appl. Toxicol. 14: 589-597, 1990) and by Pape WJW et al. (Validation of the red blood cell test system as in vitroassay for the rabbit screening of irritation potential of surfactants. Molec. Toxicol. 1: 525-536, 1987)
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-phenoxyethanol (2-PE)
- Analytical purity: ≥99.9 %

Test animals

Species:
other: rabbit, mouse, rat, dog, human (female)
Strain:
not specified
Sex:
not specified

Administration / exposure

Route of administration:
other: in vitro
Vehicle:
other: PBS
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
0, 0.5, 1, 2, 4 hours
Frequency of treatment:
single treatment
Post exposure period:
no
Doses / concentrationsopen allclose all
Dose / conc.:
20 other: mg/mL (analytical)
Remarks:
first trial
Dose / conc.:
15 other: mg/mL (analytical)
Remarks:
first trial
Dose / conc.:
7.5 other: mg/mL (analytical)
Remarks:
first trial
Dose / conc.:
3.75 other: mg/mL (analytical)
Remarks:
first trial
Dose / conc.:
1.875 other: mg/mL (analytical)
Remarks:
first trial
Dose / conc.:
0.938 other: mg/mL (analytical)
Remarks:
first trial
Dose / conc.:
15 other: mg/mL (analytical)
Remarks:
second trial
Dose / conc.:
12.5 other: mg/mL (analytical)
Remarks:
second trial
Dose / conc.:
10 other: mg/mL (analytical)
Remarks:
second trial
Dose / conc.:
7.5 other: mg/mL (analytical)
Remarks:
second trial
Dose / conc.:
5 other: mg/mL (analytical)
Remarks:
second trial
No. of animals per sex per dose:
1-3 per trial
Control animals:
yes, concurrent vehicle
Details on study design:
Reference substances:
2-Phenoxyacetic acid (2-PAA)
2-Ethoxyethanol (2-EE)
2-Ethoxyacetic acid (2-EAA)

For a human risk assessment the sensitivity of red blood cells (RBC) from mice, rats, rabbits, dogs and human to in vitro hemolysis by 2-phenoxyethanol (2-PE) was determined. Conversion of 2-PE to 2-phenoxyacetic acid (2-PAA) occurs rapidly in vivo and is considered to be an inactivation path for the hemolytic activity of 2-PE. Therefore, a comparison of the in vitro potency of the parental chemical and this metabolite was performed.
2-Ethoxyethanol (2-EE) and 2-ethoxyacetic acid (2-EAA) – chemically closely related to 2-butoxyethanol and 2-butoxyacetic acid - were also chosen for comparison. 2-EE has also been shown to be hemolytically active in rats though probably to a lesser degree than 2-PE.
The resistance of the RBC to lysis was determined with various concentrations of test compounds at different time intervals. The method employed is based on the integrity of the RBC membrane and determines the degree of cell damage and the resulting hemolysis after agitation of the cell suspensions at different test compound concentrations and time intervals. After damage of the RBC membrane by the test procedure, hemoglobin is released into the test solution. The concentration of released hemoglobin is measured, and correlated directly with damage caused to the RBC membrane by the test material.
Stock solutions of the respective test compounds were prepared in phosphate buffered saline (PBS). After pH-adjustment they were mixed in a ratio of3 volumes of test solution to 1 volume of RBC preparation to yield final test concentrations from 20 mg/ml to 0.938 mg/ml. The assay mixtures were incubated at room temperature with agitation for 0.5, 1, 2, and 4 hours, respectively. After incubation and centrifugation of the samples the release of hemoglobin into the supernatant was determined spectrometrically. Results were compared to a sample totally lysed with distilled water and to a fragility control with PBS (spontaneous, not substance related hemolysis (base line)). All samples were evaluated in triplicate.

Examinations

Examinations:
The release of hemoglobin into the supernatant as a degree of hemolysis was determined spectrometrically at a wavelength of 540 nm. Results were compared to a sample totally lysed with distilled water (100 % hemolysis) and to a fragility control with PBS (spontaneous, not substance related hemolysis (base line)). All samples were evaluated in triplicate.
Calculation of percentage hemolysis.
The percentage hemolysis produced by each test solution was calculated for each sample as follows:
The measured absorbance (A) of test solution (Test) and positive control (PC = 100 % hemolysis) were adjusted (adj.) against absorbance (A) of the fragility control (FC):
A(adj. Test) = A(Test) –A(FC)
A(adj. PC) = A(PC) – A(FC)
The adjusted absorbance of the test solution (A(adj. Test)) was then compared to the absorbance of the positive control (A(PC)) to give the percentage hemolysis (H).
H(%) = A(adj. Test) / A(adj. PC) × 100
Positive control:
Distilled water

Results and discussion

Details on results:
Total hemolysis caused by 2-phenoxyethanol (2-PE) was observed in a concentration range of 12.5 to 10.0 mg/mL in Red Blood Cell (RBC) suspensions of the different species investigated.
The results of the hemolysis tests showed the following scaling of resistance to lysis from highest to least: human > dog > rat ≈ rabbit > mouse. Human RBCs were, therefore, more resistant to 2-PE than RBCs of rabbit, dog, rat and mouse.
2-PAA, 2-EE and 2-EAA did not show significant hemolytic effects at any concentration and in any species examined.

Applicant's summary and conclusion