Phenacetin

Endpoint summary

Description of key information

Skin irritation (in vitro): Key study. Test method according to the OECD Guideline 439 with GLP study. The test item was performed with the reconstructed human SkinEthic RHE®model. The mean percent viability of the treated tissues was 93.3% versus 1.5% in the positive control. Therefore, the test item must be considered as Non-irritant to skin. 

Eye irritation (in vitro): Key study. Test method according to the OECD Guideline 438 with GLP. The test item was determined not to require 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:

15 November 2022 to 08 December 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

(SkinEthic RHE model)

Justification for test system used:

The SkinEthic RHE model has been validated for irritation testing and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE
- Tissue batch number(s): 22-RHE-149
- Delivery date:06/12/2022
- Date of initiation of testing: 06/12/2022

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no.
- Modifications to validated SOP: no.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2.
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm.
- Linear OD range of spectrophotometer: not specified.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.3 (CV=15.6%), specification OD> 0.7. Historical negative control mean OD range =0.402-1.402 (measured after a 1:2 dilution of the extracts in isopropanol).
- Barrier function: 5.4h (Specification 4.0h≤ET50≤10.0h
- Morphology: 5 cell layers
- Contamination: no.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: no interference.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg of the test item on 0.50 cm2 human skin model

Duration of treatment / exposure:

42 min at room temperature

Duration of post-treatment incubation (if applicable):

41 hours and 06 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

93.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1.5%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTABILITY CRITERIA:
- SD value of the % viability ≤18%
- Negative control: OD values of the 3 replicates in the range ≥ 0.8 and ≤ 3.0.
The optical density was measured a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥ 0.4 and ≤ 1.5.
- Positive control: Mean viability <40%.

Note:
- If the viability obtained for the test is greater than 50%, the test item has to be considered as non-irritant.
- If the viability obtained for the test item is less than or equal to 50% and the result of skin corrosion test is non-corrosive, the test item has to be considered as irritant.
- If the viability obtained for the test item is less than or equal to 50% and in absence of information on skin corrosion, the test item has to be considered as corrosive or irritant.

Table 1. Table of results

 

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	0.797	0.825	0.873	94.5	100.0	4.8	No Category
		0.848
		0.832
	SPL 2	0.889	0.900		103.1
		0.902
		0.909
	SPL 3	0.891	0.895		102.5
		0.886
		0.909
Positive control	SPL 4	0.013	0.014	0.013	1.6	1.5	0.1	Category 2 "Irritant"
		0.014
		0.015
	SPL 5	0.015	0.014		1.6
		0.013
		0.014
	SPL 6	0.014	0.012		1.4
		0.014
		0.010
Test item PH-22/0352	SPL 10	0.738	0.823	0.815	94.2	93.3	5.6	No Category
		0.875
		0.857
	SPL 11	0.747	0.762		87.3
		0.787
		0.753
	SPL 12	0.860	0.859		98.4
		0.867
		0.852

# mean of 3 values (triplicate of the same extract).

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test substance can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 93.3%

Executive summary:

An in vitro skin irritation test was conducted for the test item in a reconstructed human epidermis model (EpiDerm™) according to OECD TG 439 (GLP study). Three epidermis units, previously moistened with 10 μL of distilled water, were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated for a 41 hours and 06 minutes hours post-treatment incubation period in fresh medium at 37ºC, 5% CO2, then placed into 300 μL of fresh new assay medium during 3 hours and 45 minutes, at 37ºC, 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol.Under the test conditions, the mean percent viability of the treated tissues was 93.3%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Number of animals: Not specified.
- Characteristics of donor animals (e.g. age, sex, weight): aprox. 7 weeks old, 1.5-2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: 1h and 43 minutes
- Indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: no.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30.3-30.8 mg of test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

No post-treatment incubation is performed

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.0ºC and 32.1ºC.

After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that were not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes were rejected.

Once all eyes had been examined and approved, the eyes were incubated between 45 and 57 minutes to equilibrate them to the test system prior to dosing.

EQUILIBRATION AND BASELINE RECORDINGS
Eyes were incubated between 45 and 57 minutes to equilibrate them to the test system prior to dosing
Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED: physiological saline – Dutscher Batch No. C0866A01

SOLVENT CONTROL USED (if applicable): not applicable.

POSITIVE CONTROL USED: sodium hydroxide – Fisher Scientific, Batch No.0000080257

APPLICATION DOSE AND EXPOSURE TIME: 30.3-30.8 mg of the test item was applied for 10 seconds.

OBSERVATION PERIOD: Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: no.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points
- Damage to epithelium based on fluorescein retention: Fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
- Swelling: optical pachymeter on a slit-lamp microscope. The slit-width was set at 9 1/2 equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item
- Macroscopic morphological damage to the surface: The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.These findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean corneal swelling (%): It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula:
(corneal thickness measurement at time t - corneal thickness at time=0 / corneal thickness at ime=0 )*100
- Mean maximum opacity score:
0 - No opacity,
0.5 -Very faint opacity
1- Scattered or diffuse areas; details of the iris clearly visible
2- Easily discernible translucent area; details of the ris are slightly obscured,
3-Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4-Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment:
0-No fluorescein retention,
0.5-Very minor single cell staining,
1-Single cell staining scattered throughout the treated area of the cornea,
2-Focal or confluent dense single cell staining,
3-Confluent large areas of the cornea retaining fluorescein

Irritation parameter:

cornea opacity score

Run / experiment:

Highest mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean at 30 minutes post-treatment

Value:

1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Irritation parameter:

percent corneal swelling

Run / experiment:

All

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

morphological effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no effects

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: NO, no morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES, the combination of the three endpoints for the negative control, physiological saline, was 3 x I classified as “No Category”
- Acceptance criteria met for positive control: YES, the combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV, classified as “Corrosive/Severe Irritant ”

Table. 4: Selected eyes for the performance of the ICE test

Chamber	Fluoresceinretention	Cornealopacity	Morphological effects	Corneal thickness (e)
n°1	0.5	0	N.t.R.	0.52
n°2	0.5	0	N.t.R.	0.54
n°3	0.5	0	N.t.R.	0.50
n°4	0.5	0	N.t.R.	0.52
n°5	0.5	0	N.t.R.	0.50
n°6	0.5	0	N.t.R.	0.50
n°7	0.5	0	N.t.R.	0.54
n°8	0.5	0	N.t.R.	0.50
n°9	0.5	0	N.t.R.	0.52
n°10	0.5	0	N.t.R.	0.48
Mean corneal thickness value= Range of accepted thickness:				0.51 0.46     ≤ e ≤     0.56

N.t.R: Nothing to report

 

Table 5: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Test item

 

Endpoint measured	Eye No.	Time (min
		-45	30	75	120	180	240
Corneal opacity	4	0	0.5	0.5	0.5	0.5	0.5
	5	0	0.5	0.5	0.5	0.5	0.5
	6	0	0.5	0.5	0.5	0.5	0.5
Mean		0.0	0.5	0.5	0.5	0.5	0.5
ICE class			I
Fluorescein retention	4	0.5	1	-	-	-	-
	5	0.5	1	-	-	-	-
	6	0.5	2	-	-	-	-
Mean		0.5	1.3	-	-	-	-
ICE class			II
Corneal thickness	4	0.52	0.52	0.52	0.52	0.52	0.52
	5	0.50	0.50	0.50	0.50	0.50	0.50
	6	0.50	0.50	0.50	0.50	0.50	0.50
Corneal swelling (%)	4	0	0	0	0	0	0
	5	0	0	0	0	0	0
	6	0	0	0	0	0	0
Mean			0	0	0	0	0
ICE class			I
Combination of the 3 Endpoints		2 x I, 1 x II
CLASSIFICATION		No category

Note: No morphological effects were noted, whatever the examination time.

 

Table 6: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Positive control

 

Endpoint measured	Eye No.	Time (min)
		-45	30	75	120	180	240
Corneal opacity	1	0	4	4	4	4	4
	2	0	4	4	4	4	4
	3	0	4	4	4	4	4
Mean		0.0	4.0	4.0	4.0	4.0	4.0
ICE class			IV
Fluorescein retention	1	0.5	3	-	-	-	-
	2	0.5	3	-	-	-	-
	3	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV
Corneal thickness	1	0.46	0.70	0.82	0.84	-	-
	2	0.44	0.68	0.70	0.72	-	-
	3	0.48	0.70	0.78	0.82	-	-
Corneal swelling (%)	1	0	52	78	83	-	-
	2	0	48	52	57	-	-
	3	0	52	70	78	-	-
Mean		0.0	51	67	72	-	-
ICE class			IV
Combination of the 3 Endpoints		3 x IV
CLASSIFICATION		Category 1 : Corrosive / Severe irritant

Note:

( - ): evaluation of corneal swelling not possible (Corneal opacity = 4 at each examination time)

Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eyes No. 1, No. 2 and No. 3

 

Table 7: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Negative control

 

Endpoint measured	Eye No.	Time (min)
		-45	30	75	120	180	240
Corneal opacity	10	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class			I
Fluorescein retention	10	0.5	0.5	-	-	-	-
Mean		0.5	0.5	-	-	-	-
ICE class			I
Corneal thickness	10	0.48	0.48	0.48	0.48	0.48	0.48
Corneal swelling (%)	10	0	0	0	0	0	0
ICE class			I
Combination of the 3 Endpoints		3 x I
CLASSIFICATION		No Category

Note: No morphological effects were noted, whatever the examination time.

Interpretation of results:

other: No Category (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item was determined not to require classification for eye irritation and serious eye damage.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hidroxide (positive control) or 10μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. According to CLP Regulation EC no. 1272/2008 the test item was determined not to require classification for eye irritation and seriuous eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation/corrision: Based on the available data, the substance is considered as Non-irritant to skin according to CLP Regulation no. 1272/2008. 

Eye damage/irritation: Based on the available data, the substance does not require classification according to CLP Regulation no. 1272/2008.