3-methoxyoestra-1,3,5(10)-trien-17-one

Endpoint summary

Description of key information

In vitro skin irritation:  not irritating (Wingenroth, 2020)

in vivo skin irritation: not irritating (Kurth, 1996)

 

in vivo eye irritation: not irritating (Neckermann, 1995)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct 2020 - 15 Oct 2020

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)

Version / remarks:

2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: UN103MA
Purity: 94.98 %
Expiry date: 23 Jul 2021
Storage: room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: no data available

Source strain:

not specified

Justification for test system used:

according to guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstructed human epidermis model epiCS® (Cat.-No.CS-1001, CellSystems, Troisdorf, Germany)
- Tissue batch number(s): 100-AJ0414-1
- Date of initiation of testing: 12 Oct 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: 95%)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed control and Color control were not required.

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA:

- The mean optical density (OD) values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.


Acceptance criteria:
The following acceptance criteria determined the validity of an assay:
- mean OD negative control >/=0.8 and - mean relative viability of the positive control is - If the mean viability of the 3 replicates > 20% the coefficient of variation (CV) should not exceed 0.3.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

105.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Direct-MTT reduction: no
- Colour interference with MTT: no

- DEMONSTRATION OF TECHNICAL PROFICIENCY:
Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Conclusions:

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 105.6%. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

Executive summary:

A study was performed for the assessment of the skin irritancy of Estron methylether with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.

The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.
Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100% The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.
The mean value of cell viability was recorded to be 105.6%. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13. July to 14. Aug 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Amendment to Annex VI of the Directive 67/548 EEC in the version of EEC Directive 93/21 EEC and "Gefahrstoffverordnung, Stand Oct. 94"

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

combined acute toxicity and local tolerance study

Deviations:

yes

Remarks:

24 h exposure

Principles of method if other than guideline:

A combined study on acute dermal toxicity and on local tolerance was used to assess skin irritation/corrosion (comparable to OECD TG 402 and 404). The study design and reporting deviates from the current OECD TG 404, however, the study is considered sufficient for assessment of this endpoint. As described in ATP 96/54/EC ANNEX IV A, PART B: METHODS FOR THE DETERMINATION OF TOXICITY AND OTHER HEALTH EFFECTS, a strategy of testing for irritancy allows the non-performance of a test if an acute toxicity study by the dermal route has been conducted at the limit test dose with the substance (method B.3), and no skin irritation was observed, further testing for skin irritation (method B.4) may be unnecessary.

GLP compliance:

yes

Specific details on test material used for the study:

- stability: The formulation was prepared fresh on application day and the administrations were carried out within approximately 3.0 hours.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Schering AG
- Weight at study initiation: males: 109-115 g; females: 96-107 g
- Fasting period before study: ca. 19 hours
- Housing: 1/cage
- Diet (e.g. ad libitum): pell. Altromin R, ad libitum
- Water (e.g. ad libitum): demineralized acidified water, pH 2-3, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-24°C
- Humidity (%): 42-60%
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

physiological saline

Remarks:

900 mg NaCI + 85 mg Myrj® 53 ad 100 ml bidist. water

Controls:

no

Amount / concentration applied:

2000 mg/kg bw

Duration of treatment / exposure:

24 h

Observation period:

14 days

Number of animals:

3/sex

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: start (day 1), on day 7 and at the end (day 14) of the study
- Necropsy of survivors performed: yes
- evaluation of local tolerance 24, 48 and 72 hours after the end of administration

Irritation parameter:

erythema score

Basis:

animal: 3 females

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

erythema score

Basis:

animal: 3 males

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

animal: 3 female

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

animal: 3 males

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Other effects:

none

Interpretation of results:

GHS criteria not met

Conclusions:

Estrone-methyether did not show a skin irritating or corrosive potential in rats when administered occlusively to the shorn back for 24 hours.

Executive summary:

In a combined study on acute toxicity and on local tolerance similar to OECD TG 404 Wistar rats (3/sex) (3/sex) were dermally exposed to Estrone-methyether  in physiological saline for 24 hours at a limit dose of 2000 mg/kg bw.  Animals then were observed for 14 days.

The administration of the test substance was tolerated without mortalities, compound-related clinical findings, effects on body weight gain and gross pathological findings.

No local intolerance reactions at the application sites were observed.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Schriever
- Weight at study initiation: 3.6-4.6 kg
- Housing: 1/cage
- Diet (e.g. ad libitum): pell. Altromin® K, ad libitum
- Water (e.g. ad libitum): demineralized water, ad libitum
- Acclimation period: > 14 days; during this period a diet containing 0.03% sulfaquinoxaline was offered for 7 consecutive days as coccidiosis prophylaxis; the animals received Jacutin® emulsion into the meatus acusticus externus for prophylaxis against mite infection of the ear and they were vaccinated against rabbit haemorrhagic disease (Arvilap®)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22°C
- Humidity (%): 56-62%
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated left eye served as control

Amount / concentration applied:

0.1 ml/eye (approx. 63-72 mg)

Duration of treatment / exposure:

test termination day 4, test substance remained in eye

Observation period (in vivo):

until day 4

Number of animals or in vitro replicates:

4 females

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Other effects:

no local findings were observed in the treated and untreated eyes

Interpretation of results:

GHS criteria not met

Conclusions:

non-eye irritant

4 rabbits were treated with estrone-methylether in the right eye.

The test substance did not show any eye irritating potential after single conjunctival application.

Executive summary:

In a primary eye irritation study according to OECD TG 405 0.1 ml Estrone-methylether (63-72 mg) was instilled into the conjunctival sac of the right eye of New Zealand White rabbits (4 f). The untreated left eye served as control. Animals were observed for 4 d. Conjunctivae, eyelids, cornea and iris were evaluated before application, 0.5, 1 and 2 hours thereafter, and then once daily until termination of the study on day 4.

The test item was tolerated without causing local findings. No animal reacted positively according to the scoring system. The control eyes were without findings. In this study, Estrone-methylether was not irritating to the eye.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

With respect to local irritant effects, an in vitro test for skin irritation/corrosion (OECD TG 439) and an in vivo study investigating damage to the eye (OECD TG 405) is available for estrone-3-methylether. As supporting information, the combined acute dermal toxicity and local tolerance study of estrone-3-methylether in rats is used.

 

Skin irritation

A study was performed for the assessment of the skin irritancy of Estron methylether with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.

The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.
Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100% The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.
The mean value of cell viability was recorded to be 105.6%. The test item was thus shown to be not irritating to reconstructed human skin in vitro (Wingenroth, 2020).

 

In a combined study on acute toxicity and on local tolerance similar to OECD TG 404 Wistar rats (3/sex) (3/sex) were dermally exposed to Estrone-methyether in physiological saline for 24 hours at a limit dose of 2000 mg/kg bw.  Animals then were observed for 14 days.

The administration of the test substance was tolerated without mortalities, compound-related clinical findings, effects on body weight gain and gross pathological findings.

No local intolerance reactions at the application sites were observed (Kurth, 1996).

 

Supporting data are available for the source substance estrone:

A study was performed for the assessment of the skin irritancy of Estrone with a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)). The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.

The skin tissue was moistened with 5 μl of Milli-Q water and 10.1 mg of Estrone was applied undiluted topically to the skin tissue construct in triplicates and incubated for 15 minutes, followed by a 42 hours post-treatment incubation period.
Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%. The results of the concurrent negative control (NC, PBS) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.
The relative mean tissue viability obtained after 15 minutes treatment with Estrone compared to the negative control tissues was 99%. Since the mean relative tissue viability for Estrone was above 50% after 15 minutes treatment Estrone is considered to be non-irritant.

The positive control had a mean cell viability of 12% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 12%, indicating that the test system functioned properly (Verbaan, 2011).

 

Eye irritation

In a primary eye irritation study according to OECD TG 405 0.1 ml Estrone-methylether (63-72 mg) was instilled into the conjunctival sac of the right eye of New Zealand White rabbits (4 f). The untreated left eye served as control. Animals were observed for 4 d. Conjunctivae, eyelids, cornea and iris were evaluated before application, 0.5, 1 and 2 hours thereafter, and then once daily until termination of the study on day 4.

The test item was tolerated without causing local findings. No animal reacted positively according to the scoring system. The control eyes were without findings. In this study, Estrone-methylether was not irritating to the eye (Neckermann, 1995).  

 

Supporting data are available for the source substance estrone:

 

A study was performed to assess the eye irritation potential of the test item Estrone with an in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic membrane (CAM) of fertilized chicken eggs.

After the application of the test item blood vessels and albumen were examined and scored for the following irritant effects: vasodilation, slight haemorrhage; vessel lysis, strong haemorrhage; blood coagulation, albumen coagulation for a period of 300 seconds.

A volume of 300 μl of the test item (corresponding to an average of 190 mg) was applied per egg directly onto the chorioallantoic membrane.

The Irritation Score (IS) value was calculated to be 0 for the test item which was interpreted as being non-irritant. The results of the positive (SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system (Wingenroth, 2016).

 

An in vitro study for assessing ocular irritation of compounds was performed with the test item Estrone using a human epithelial corneal cell model. The model used is standardized and commercially available (Human Corneal Epithelial Model (HCE); Episkin, France). The study was conducted equivalent to the later implemented OECD TG 492.

Undiluted Estrone was applied topically to the HCE tissue, i.e. 30 mg per insert; plus 30 μl PBS to moisten and ensure good contact with the tissue; three replicates.

After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 98% (rounded) as measured by a MTT conversion assay.

The results of the concurrent negative (NC, PBS) and positive control (PC, SDS 0.3 %) demonstrated the viability (NC) and sensitivity (PC) of the test model.

The results show that Estrone is predicted as non-irritant under the conditions of this test method (Wingenroth, 2016).

 

A study was performed to assess the corneal damage potential of the solid test item Estrone with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea in accordance with OECD TG 437.

The possible ocular irritancy of Estrone was tested through topical application for 240 ± 10 minutes.

The test item was a white powder with a purity of >95%. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (302 to 334 mg).

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% w/v Imidazole) was 106 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Estrone did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.8 after 240 minutes of treatment.

Finally, it is concluded that Estrone is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report (Verspeek-Rip, 2011).

 

Based on these assays no corrosive or irritant potential to the skin and no damage to the eye can be concluded for estrone-3-methylether.

These findings are supported by in vitro data for estrone used as source for read-across in other sections of this document. No skin irritation (OECD TG 439) and no eye irritation (OECD TG 437) was found for estrone used with a purity of >95% supported by two further in vitro studies (HCE and HET-CAM, substance purity >98% in both studies) with no findings (Table 11).

 

Klimisch reliability score 1 or 2 is concluded for the in vitro studies with estrone and estrone-3-methylether and reliability score 2 is concluded for the in vivo studies (due to limited scope of report) with estrone-3-methylether.

In conclusion, estrone-3-methylether is not classified (Reg. (EC) No. 1272/2008) to induce skin irritation/corrosion or damage to the eye.

 

Skin and eye irritation/ corrosion studies for estrone and estrone-3-methylether

	Estrone	Estrone-3-methylether
CAS No.	53-16-7	1624-62-0
ZK	5019	5512
Skin irritation (RhE) in vitro OECD TG 439	NOTOX project 495753 (Verbaan, 2011), EpiSkin-SM GLP, guideline study, no deviations Batch no. EFB465K1A; purity >95% No findings -> Not irritant (rel-1)	Study no. T105014-1/ Report no. AT07456 (Wingenroth, 2020), epiCS GLP, guideline study, no deviations Batch no UN103MA; purity 94.98% No findings -> Not irritant (rel-1)
Skin irr. in vivo (rat) comparable to OECD TG 404	No data	Comb. acute dermal & local tolerance study; TX95190/ X052; draft (Kurth, 1996) GLP, comparable to TG 404 with deviations; please refer to 4.3 Batch no. 11427106; purity 96% No findings -> not irritant (rel-2)
BCOP Eye corr./ irrit. in vitro OECD TG 437	NOTOX project 495754 (Verspeek-Rip, 2011), GLP, guideline study, Batch no. EFB465K1A; purity >95% No findings -> Not corrosive (rel-1)	No data
HCE Eye irrit. In vitro equivalent OECD TG 492	Study no. T102260-1/ Report no. PH-39215 (Wingenroth, 2016), HCE- EpiSkin, GLP, (pre-guideline study) equivalent to TG 492, deviation of positive control (SDS) Batch no 13578002; purity 98.5% No findings -> Not irritant (rel-1)	No data
HET-CAM Eye irrit. in vitro ICCVAM Test Method	Study no. T102261-2/ Report no. PH-39216 (Wingenroth, 2016), GLP, HET-CAM, equivalent to guideline study, Batch no 13578002; purity 98.5% No findings -> Not irritant (rel-1)	No data
Eye corr. in vivo (rabbit), OECD TG 405	No data	TX95217/ X048; draft (Neckermann, 1995); GLP, equivalent to guideline (version 1987) Batch no. 11427106; purity 96.1% Limited scope of report, focus on conjunctiva, no individual animal reporting; sufficient for assessment No findings -> Not irritant (rel-2)

 

 

Justification for classification or non-classification

Based on the available data no classification for írritation/corrosion (skin and eye) according to Regulation (EC) No. 1272/2008 (CLP) is required.