Estradiol

Endpoint summary

Description of key information

study report: OECD 406, not sensitizing [Hart, 1989]

study report: OECD 406, not sensitizing [Hart, 1989b]

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1989 to December 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

A valid Buehler test conducted comparable to guideline with acceptable restrictions is available, which is reliable with restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, only a minor indication for skin sensitisation was observed in this study, presumably rather based on the adhesive used in the patch and not due to the test substance, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

not specified

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, MA
- Diet (e.g. ad libitum): Ad libitum, Ralston Purina Guinea Pig Chow, Ralston Purina, St. Louis, MO
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Several days
- Indication of any skin lesions: No

Route:

intradermal

Vehicle:

water

Remarks:

Freund’s Adjuvant (1:1 commercial adjuvant with water) in an area over which the test article was immediately applied

Concentration / amount:

Freund’s Adjuvant (1:1 commercial adjuvant with water) in an area over which the test article was immediately applied

Day(s)/duration:

two injections with 0.1 mL of solution prior to topical application

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

5 cm² patch containing approx. 1.5 mg/patch

Day(s)/duration:

three application per week (Monday, Wednesday, and Friday)/ eight topical applications in total

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Remarks:

Estradiol TDD patch

Concentration / amount:

approx. 1.5 mg/patch

Day(s)/duration:

applied 14 days after last induction and with a duration of 24 h.

No. of animals per dose:

10

Details on study design:

RANGE FINDING TESTS: Due to the nature of the test article (an adhesive patch) and the
positive response of two animals during the challenge phase of the sensitization study, a guinea pig primary skin irritation study (5 animals) was conducted. The minimal irritation noted during the primary skin study suggests the erythema and edema noted during the challenge phase may have been due to irritation.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 8
- Exposure period: every second day
- Test groups: 1
- Control group: 1
- Site: dorsal shoulder girdle
- Frequency of applications: every second day (Monday, Wednesday, and Friday)
- Duration: 6h each
- Concentrations: approx. 1.5 mg/patch

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 14 days after last induction
- Exposure period: 24h
- Test groups: 1
- Control group: 1
- Site: dorsal shoulder girdle
- Concentrations: 1.5 mg/patch
- Evaluation (hr after challenge): 24 and 48 h

Shortly before the initial topical application two intradermal injections with Freund´s adjuvant were conducted in the same area at which the patches were placed.

Positive control substance(s):

yes

Remarks:

2,4-Dinitrochlorobenzene (DNCB)

Positive control results:

Sensitization: 100%

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Remarks on result:

other: the performance of a negative control was not reported

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Remarks on result:

other: the performance of a negative control was not reported

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.05% DNCB

No. with + reactions:

10

Total no. in group:

10

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1.5 mg/patch

No. with + reactions:

1

Total no. in group:

10

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.05% DNCB

No. with + reactions:

10

Total no. in group:

10

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1.5 mg/patch

No. with + reactions:

2

Total no. in group:

10

Primary Skin Irritation test

	Irritation scores for abraded skin sites after removal
	One hour		Day 2
Animal Number	Erythema	Edema	Erythema	Edema
9G2929	1E	0	0	0
9G2934	0	0	0	0
9G2960	0	0	0	0
9G2965	1E	0	0	0
9G2930	0	0	0	0
Mean	0.4	0.0	0.0	0.0
Subtotal		0.4
Key	E= Epithelial Stripping

Results Test item

	One day		Two days
Animal Number	Erythema	Edema	Erythema	Edema
9G2907	0	0	0	0
9G2912	0	0	0	0
9G2917	0	0	0	0
9G2922	1E	0	0	0
9G2908	1	0	1	0
9G2913	0	0	0	0
9G2918	0	0	0	0
9G2923	0	0	0	0
9G2909	0	0	0	0
9G2914	0	0	0	0

Results Positive Control

	One day		Two days
Animal Number	Erythema	Edema	Erythema	Edema
9G2957	1	0	1	0
9G2962	1	0	0	0
9G2927	1	0	2	0
9G2932	1	1	1	0
9G2958	1	0	2	0
9G2963	2	0	2	1
9G2928	1	1	1	1
9G2933	1	0	1	0
9G2959	1	0	2	0
9G2964	2	0	2	0

 

 

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

In the present test conducted similar to OECD 406 (1981); Bühler method, 10 Dunkin-Hartley guinea pigs were dermally exposed to Estradiol via TDD Patches containing approximately 1.5 mg Estradiol as indicated in the local tolerance test. The animals were exposed eight times every second day, prior to the initial application an intradermal application of Freund´s adjuvant was performed. Fourteen days after the last topical application the animals were again dermally exposed to the patches for 24h and the reactions were documented after 24h and 48h. Only slight Erythema nor Edema occurred after induction or challenge with the Estradiol TDD patches. Erythema was scored with 1 in two animals, in one animal the irritation was found to be due to the used adhesive of the patches causing epithelial stripping. In the second animal Erythema lasted until the second reading. According to CLP, EU GHS (Regulation (EC) No 1272/2008), a response of at least 30% of the test animals of an adjuvant type guinea pig test method for skin sensitisation is considered as positive. thus the substance does not meet the classification criteria for skin sensitization according to Regulation (EU) No. 1272/2008 (CLP) or the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Executive summary:

In a dermal sensitisation study comparable to OECD guideline 406 (1981) with Estradiol TDD patches 10 young adult Hartley guinea pigs were tested using the method of Buehler.

Test concentrations were selected based on the results of an irritation screening study: the concentrations used in the pretest (approximately 1.5 mg/patch of the test substance) produced only a minor irritation in one animal. Thus, in the main study 1.5 mg of the test substance contained in the TDD patch were used for epicutaneous induction and challenge. Simultaneously to the initial topical induction an intradermal injection with Freund´s adjuvant was performed.

After challenge only minor visible changes of the treated skin sites were observed in the test group animals 24 and 48 h after patch removal (= grade 0.4).

The positive control DNCB produced a sensitisation rate of 100%.

The test material produced a response in 20% of animals. According to CLP, EU GHS (Regulation (EC) No 1272/2008), a response of at least 30% of the test animals of an adjuvant type guinea pig test method for skin sensitisation is considered as positive.

Estradiol is not a dermal sensitiser in this study.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January to March 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

A valid Buehler test conducted comparable to guideline with acceptable restrictions is available, which is reliable with restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

not specified

Details on test animals and environmental conditions:

TEST ANIMALS
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: several days
- Indication of any skin lesions: No

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.1 mL Freund´s adjuvant (1:1 commercial adjuvant with water)

Day(s)/duration:

Two injections prior to initial topical application at the site over which the test article was immediately applied.

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

patches containing estradiol

Day(s)/duration:

nine topical applications for 6 to 7 h on three applications days per week: Monday, Wednesday and Friday

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

patches containing estradiol

Day(s)/duration:

24 h 14 days after induction phase

No. of animals per dose:

10

Details on study design:

RANGE FINDING TESTS: not reported

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 9
- Exposure period: every second day
- Test groups: 1
- Control group: 1
- Site: dorsal shoulder girdle
- Frequency of applications: every second day
- Duration: 6 to 7 h
- Concentrations: not reported ready to use product, approximately 1.5 mg per patch as indicated in the local tolerance study

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 24 h
- Exposure period: 24 h
- Test groups: 1
- Control group: 1
- Site: dorsal shoulder girdle
- Concentrations: not reported ready to use product, approximately 1.5 mg per patch as indicated in the local tolerance study
- Evaluation (hr after challenge): 24 and 48 h

Positive control substance(s):

yes

Remarks:

2,4-dinitrochlorobenzene (DNCB)

Positive control results:

Sensitization: 100%

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Remarks on result:

other: the performance of a negative control was not reported

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Remarks on result:

other: the performance of a negative control was not reported

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.01% DNCB

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

approx. 1.5 mg/patch

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.01% DNCB

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

approx. 1.5 mg/patch

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results for Patch application

	One day		Two days
Animal Number	Erythema	Edema	Erythema	Edema
9G122	0	0	0	0
9G128	0	0	0	0
9G134	0	0	0	0
9G140	0	0	0	0
9G146	0	0	0	0
9G123	0	0	0	0
9G129	0	0	0	0
9G135	0	0	0	0
9G141	0	0	0	0
9G147	0	0	0	0

Results for positive control (DNCB)

	One day		Two days
Animal Number	Erythema	Edema	Erythema	Edema
9G124	1	0	1	0
9G130	1	0	1	0
9G136	1	0	1	0
9G142	1	0	2	0
9G148	2	0	2	0
9G125	2	0	2	0
9G131	2	0	2	0
9G137	1	0	1	0
9G143	1	0	2	0
9G149	2	0	2	0

 

 

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

In the present test conducted similar to OECD 406 (1981); Bühler method, 10 Dunkin-Hartley guinea pigs were dermally exposed to Estradiol via TDD Patches containing approximately 1.5 mg Estradiol as indicated in the local tolerance test. The animals were exposed nine times every third day, prior to the initial application an intradermal application of Freund´s adjuvant was performed. Fourteen days after the last topical application the animals were again dermally exposed to the patches for 24h and the reactions were documented after 24h and 48h. No Erythema nor Edema occurred after induction or challenge with the Estradiol TDD patches, thus the substance does not meet the classification criteria for skin sensitization according to Regulation (EU) No. 1272/2008 (CLP) or the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Executive summary:

In a dermal sensitisation study comparable to OECD guideline 406 (1981) with Estradiol TDD patches 10 young adult Hartley guinea pigs were tested using the method of Buehler.

Test concentrations were selected based on the results of an irritation screening study: the concentrations used in the pretest (approximately 1.5 mg/patch of the test substance) produced only a minor irritation in one animal. Thus, in the main study 1.5 mg of the test substance contained in the TDD patch were used for epicutaneous induction and challenge. Simultaneously to the initial topical induction an intradermal injection with Freund´s adjuvant was performed.

After challenge no visible changes of the treated skin sites were observed in the test  group animals 24 and 48 h after patch removal (= grade 0).

The positive control DNCB produced a sensitisation rate of 100%.

The test material produced a response in 0% of animals. According to CLP, EU GHS (Regulation (EC) No 1272/2008), a response of at least 30% of the test animals of an adjuvant type guinea pig test method for skin sensitisation is considered as positive.

Estradiol is not a dermal sensitiser in this study.

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Estradiol is an endogenous human sex hormone and an approved drug since several decades. Therefore, human data are available for the assessment of the skin sensitization potential of estradiol and the related substances. The most common route of administration is by oral intake or transdermal with patches delivering the respective hormone, e.g. 17ß-estradiol. For the assessment of the skin sensitization potential the experience with the transdermal delivery systems is most relevant. The main principle of a transdermal therapeutic system is a drug reservoir or matrix, an occlusive backing, a membrane that allow controlled diffusion of the drug to the skin, into and through the skin (systemic availability and therapeutic principle) and an adhesive for attachment to the skin (for overview see e.g. Chetkowski et al. 1986, Wiseman & Travish 1994, Alberti et al. 2005, Nath & Sitruk-Ware 2009).

 

Human data can be distinguished in experimental data, clinical trials, and case studies.

 

In clinical case studies the main focus is on adverse effects of the treatment and the application form, whereas the experimental data and the clinical trials have a broader basis, i.e., a higher number of participants and thus, give more reliable information about the effects of the substance.

 

However, in the review of Stropp (2016) from the case studies only 17 cases could be identified where estradiol was applied either dermally via a transdermal therapeutic system (TTS), as gel, dissolved in ethanol or given orally and was subsequently correlated with irritation effects or positive sensitization responses.

 

In contrast, in the clinical trials studies several hundred patients were treated with estradiol over a period of several weeks to years and overall, a good tolerability was reported. Also, in this clinical trails skin irritation occurs but mainly only to some time-points in the whole study period and not severe enough to quit the treatment.

 

E.g., in a randomized study examined the continuous combined transdermal delivery of estradiol alone or estradiol combined with norethisterone acetate with different concentrations across 37 centers in the USA. The patches were applied every 3.5 days and the study duration was 52 weeks. 625 patients were treated with respective patches in four groups. Erythema was the most frequent application site reaction, reported at least once during the 52 weeks (Archer et al. 1999). It is assumed that the application site reaction is related to irritation due to the patch and not evidence of a skin sensitization reaction, as effects were observed sporadically (“at least once during...”) and did not lead to a discontinuation of the treatment.

 

In a 12 week, double-blind, placebo-controlled and multicenter trial 196 menopausal women were either treated with transdermal continuous estradiol at different doses or placebo. Good local skin tolerance was reported, and effects at the application site were recorded in 4.9 to 10.7% of the patients in the estradiol groups and in 7.4% of the placebo group. It was concluded that the occurrence of skin reactions may be related to the size of the patch (Utian et al. 1999). In this study there is no evidence of a skin sensitization reaction related to estradiol, and the skin irritation effects observed are assumed to be related to the size of the transdermal delivery system.

 

In a review from 1994 it was concluded from earlier studies that “the combined estradiol/norethisterone transdermal delivery system appeared to cause a similar incidence of skin reactions as that observed with estradiol-only patch in clinical trials with more than 400 patients” and that “Skin reactions can be minimised by selecting new sites when applying the systems, and by application to the buttocks rather than the abdomen or lower back” (Wiseman & McTavish 1994). Therefore, also from this review there is no evidence of a skin sensitization reaction related to estradiol or the transdermal delivery system.

 

 

Similarly, in the experimental studies a skin sensitizing potential was observed which was not considered estradiol related.

 

An experimental study (modified Draize-Shelanski-Jordan method) on two estradiol containing transdermal therapeutic systems was done in 24 healthy women. A reservoir-type transdermal system and a matrix-type transdermal system was examined in 12 women each with an induction period over three and a half week (7 applications) and a challenge treatment after a 17-day rest phase. Whereas some responses were observed during induction, no clinical signs or subjective symptoms occurred during the challenge phase. Therefore, there was no evidence of a skin sensitization potential under the test conditions used (Rozenbaum et al. 1996). In an earlier publication it was reported that an investigation in 25 volunteers “found that no subjects developed contact allergy from using the estrogen patch” (no further details given; Utian 1987).

 

In addition to the above mentioned clinical trails, case studies and human experimental data animal studies were also conducted with Estradiol.

Estradiol was examined in two non-mouse local lymph nodes assays in 1989 (Hart 1989; Hart 1989b). In both studies a transdermal therapeutic system was use for the application of estradiol in a concentration of approx. 1.5 mg. The experiments were conducted according to OECD test guideline 406 (Bühler method). In one of both assays slight erythema were observed in two of ten animals at the first reading point (24h) and in one of ten animals at the second reading point (48h). Thus, based on the classification criteria < 30% of the animals exhibited a reaction after the application of the test item in a TTS and thus, the test item is not considered to be a dermal sensitizer. In the other study none of the animals exposed to the test substance showed any erythema nor edema at the reading points 24 and 48h after treatment. In both assays the test item is not considered to be a dermal sensitizer under the conditions of the test.

In silico evaluation of a sensitizing potential:

 

Estradiol was profiled for a sensitizing potential using the OECD QSAR Toolbox.

The “Profiling” module contains all the knowledge in the system coded in profiling schemes (profilers). The profilers identify the affiliation of the target chemical(s) to preliminary defined categories (functional groups/alerts).

The outcome of the profiling determines the most appropriate way to search for analogues, but they are also useful for preliminary screening or prioritization of substances.

The information provided is the structure characterization (functional groups, chemical elements, etc.) of the target or known mechanisms of action (alerts) associated with a specific functionality in the target structure. A mechanistic justification for the identified alerts is provided.

 

Estradiol (CAS: 50-28-2)-profiling results for sensitization from OECD QSAR Toolbox 4.4.1:

	Profilers
	Predefined
	General Mechanistic
(1)	Protein binding by OECD	No alert found
(2)	Protein binding by OASIS	No alert found
(3)	Protein binding potency Lys (DPRA 13%)	DPRA less than 9% (DPRA 13%) DPRA less than 9% (DPRA 13%) >> Alcohols
(4)	Protein binding potency Cys (DPRA 13%)	DPRA less than 9% (DPRA 13%) DPRA less than 9% (DPRA 13%) >> Alcohols
(5)	Protein binding potency GSH	Not possible to classify according to these rules (GSH)
	Endpoint Specific
(6)	Protein binding alerts for skin sensitization according to GHS	No alert found
(7)	Protein binding alerts for skin sensitization by OASIS	No alert found
(8)	Protein Binding Potency h-CLAT	No alert found
(9)	Keratinocyte gene expression	Not possible to classify according to these rules
(10)	Respiratory sensitisation	No alert found
	Custom
(11)	Skin sensitisation for DASS	Positive

 

The profilers and their development are explained below:

• Protein binding by OECD:

The Protein binding by OECD profiler was developed by an analysis of direct acting structural alerts based on theoretical organic chemistry (the profiler does not contain metabolically / abiotically activated structural alerts). The alert compilations were analysed in order to place the information contained within the literature into a mechanistic chemistry framework. This mechanistic chemistry can be used as the basis for chemical category formation when utilising the Protein binding by OECD profiler. Within each of the five mechanistic domains related structural alerts have been grouped based on the presence of a common reactivity site into so-called mechanistic alerts. Chemical category formation can be carried out at either the mechanistic alert or structural alert level using this profiler. The protein binding by OECD profiler contains 16 mechanistic alerts covering 52 structural alerts. These data are supported by mechanistic chemistry and references to the scientific literature (the meta data).

Relevance/Applicability to endpoint(s)

This profiler is intended to be used for the assessment of endpoints in which covalent binding to a protein has been shown to be the molecular initiating event for low molecular weight chemicals. The profiler has been developed from mechanistic knowledge of the electrophilic chemistry of covalent protein binding for direct acting electrophiles only – importantly it has been developed from a systematic review of the literature and not from the analysis of a single toxicological dataset.

Relevance/Applicability to particular chemical classes

This profiler is applicable only to organic chemicals that have a molecular weight less than 1000 g/mol. It is applicable only to the chemical classes for which it contains structural alerts; the absence of a structural alert should not be taken as an absence of toxicity. This profiler contains structural alerts for direct acting electrophiles only – oxidation and/or metabolism are not accounted for (appropriate Toolbox simulators should be applied, if required).

 

(2): Protein binding by OASIS

The scope of the profiler is to investigate presence of alerts within target molecules responsible for interaction with proteins. The list of 112 structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

Relevance/Applicability to endpoint(s)

The profiler is based on the rules defined in the OASIS TIMES models for Skin sensitisation (SS). It consists of 112 structural alerts related to interactions with proteins especially skin proteins and proteins such as topoisomerases, cellular protein adducts, etc. It is believed that positive results are result of interactions with proteins. The list of structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

Relevance/Applicability to particular chemical classes

This profiler is applicable to those organic chemicals that have presence of at least one of the 112 protein binding alerts specified within the profiler. The presence of protein binding alerts is not bounded with parametric ranges; it is based on structural boundaries only.

(3) Protein binding potency Lys (DPRA 13%):

This profile is built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization. It is developed on the base of data derived from Direct Peptide Reactivity Assay (DPRA). The DPRA is a reactivity assay which evaluates the ability of chemicals to react with proteins. As model peptides are used reduced glutathione and two synthetic peptides – lysine and cysteine. The reaction time for both lysine and cysteine is 24 hours. The peptide reactivity is reported as percent peptide depletion. The profile contains 73 structural alerts extracted from about 228 chemicals with experimentally measured lysine depletion values. The set of 73 structural alerts are separated into three potency categories: DPRA above 21% (DPRA 13%), DPRA less than 9% (DPRA 13%) and Grey zone 9-21% (DPRA 13%). Classification of potency categories is based on analysis published in a collaboration with L`Oreal (Dimitrov et al., 2016).

The data used to derive the structural boundaries have been taken by three literature sources: Natsch et al., 2015; Urbisch et al., 2015 and Jaworska et al., 2015 Gerberick, G.F., Vassallo, J.D., Bailey, R.E., Chaney, J.G., Morrall, S.W. and Lepoittevin, J.P. 2004.

Development of a peptide reactivity assay for screening contact allergens. Toxicol. Sci. 81: 332-343. Natsch, A. and Gfeller, H. 2008.

LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitisation potential. Toxicol. Sci. 106: 464-478. Natsch, A., Emter, R., Gfeller, H., Haupt, T. and Ellis, G. 2015.

Predicting skin sensitizer potency based on in vitro data from KeratinoSens and kinetic peptide binding: global versus domain-based assessment. Toxicol. Sci. 143(2), 319-332. Jaworska, J., Natsch, A., Ryan, C., Strickland, J., Ashikaga, T., Miyazawa, M. 2015.

Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy. Arch Toxicol, 2355-2383. Urbisch, D., Mehling, A., Guth, K., Ramirez, T., Honarvar, N., Kolle, S., Landsiedel, R., Jaworska, J., Kern, P., Gerberick, F., Natsch, A., Emter, R., Ashikaga, T., Miyazawa, M., Sakaguchi, H. 2015.

Assessing skin sensitization hazard in mice and men using non-animal test methods. Regulatory Toxicology and Pharmacology 71: 337-351. S. Dimitrov, A. Detroyer, C. Piroird, C. Gomes, J. Eilstein, T. Pauloin, C. Kuseva, H. Ivanova, I. Popova, Y. karakolev, S. Ringeisses, O. Mekenyan, Accounting for data variability, a key factor in in vivo/in vitro relationships: application to the skin sensitization potency (in vivo LLNA versus in vitro DPRA) example. J Appl Toxicol, 2016,  DOI 10.1002/jat.3318

Relevance/Applicability to particular chemical classes

This profiler is applicable to those organic chemicals that have presence of a functional group reacting with the lysine residue. The presence of an alert is not bounded with parametric ranges; it is based on structural boundaries only.

 

(4) Protein binding potency Cys (DPRA 13%):

This profile is built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization. It is developed on the base of data derived from Direct Peptide Reactivity Assay (DPRA). The DPRA is a reactivity assay which evaluates the ability of chemicals to react with proteins. As model peptides are used reduced glutathione and two synthetic peptides – lysine and cysteine. The reaction time for both lysine and cysteine is 24 hours. The peptide reactivity is reported as percent peptide depletion. The profile contains 77 structural alerts extracted from about 229 chemicals with experimentally measured cysteine depletion values. The set of 77 structural alerts are separated into three potency categories: DPRA above 21% (DPRA 13%), DPRA less than 9% (DPRA 13%) and Grey zone 9-21% (DPRA 13%). Classification of potency categories is based on analysis published in a collaboration with L`Oreal (Dimitrov et al., 2016).

The data used to derive the structural boundaries have been taken by three literature sources: Natsch et al., 2015; Urbisch et al., 2015 and Jaworska et al., 2015 Gerberick, G.F., Vassallo, J.D., Bailey, R.E., Chaney, J.G., Morrall, S.W. and Lepoittevin, J.P. 2004. Development of a peptide reactivity assay for screening contact allergens. Toxicol. Sci. 81: 332-343.

Natsch, A. and Gfeller, H. 2008. LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitisation potential. Toxicol. Sci. 106: 464-478.

Natsch, A., Emter, R., Gfeller, H., Haupt, T. and Ellis, G. 2015. Predicting skin sensitizer potency based on in vitro data from KeratinoSens and kinetic peptide binding: global versus domain-based assessment. Toxicol. Sci. 143(2), 319-332.

Jaworska, J., Natsch, A., Ryan, C., Strickland, J., Ashikaga, T., Miyazawa, M. 2015. Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy. Arch Toxicol, 2355-2383.

Urbisch, D., Mehling, A., Guth, K., Ramirez, T., Honarvar, N., Kolle, S., Landsiedel, R., Jaworska, J., Kern, P., Gerberick, F., Natsch, A., Emter, R., Ashikaga, T., Miyazawa, M., Sakaguchi, H. 2015. Assessing skin sensitization hazard in mice and men using non-animal test methods. Regulatory Toxicology and Pharmacology 71: 337-351.

10. Dimitrov, A. Detroyer, C. Piroird, C. Gomes, J. Eilstein, T. Pauloin, C. Kuseva, H. Ivanova, I. Popova, Y. karakolev, S. Ringeisses, O. Mekenyan, Accounting for data variability, a key factor in in vivo/in vitro relationships: application to the skin sensitization potency (in vivo LLNA versus in vitro DPRA) example. J Appl Toxicol, 2016, DOI 10.1002/jat.3318

Relevance/Applicability to particular chemical classes

This profiler is applicable to those organic chemicals that have presence of a functional group reacting with the cysteine residue. The presence of an alert is not bounded with parametric ranges; it is based on structural boundaries only.

 

(5) Protein binding potency GSH:

This profiler is developed on the base of empirical data for thiol reactivity expressed by the in chemico RC50 value. Data are obtained by measuring target chemical covalent binding with the thiol group of glutathione (GSH). The structural alerts for protein binding are extracted from about 1455 chemicals comprised within GSH Experimental RC50. All the chemicals have two common electrophilic mechanisms of interaction with GSH – interaction via SN2 and interaction via Michael addition (MA) mechanism.

The profiler contains 137 categories structural alerts which are separated into five potency categories: Extremely, Highly, Moderately, Slightly reactive and Suspect.  Classification of potency categories is as follows: extremely reactive (RC50 < 0.099mmol/L); highly reactive (RC50 = 0.100 – 0.990mmol/L); moderately reactive (RC50 = 1.000 – 15.000mmol/L); slightly reactive (RC50 = 16.000 – 70.000mmol/L); suspect (RC50 = 71.000 – 135.000).

The profiling results outcome assigns a target to the corresponding potency category based on matched structural criteria.

Relevance/Applicability to particular chemical classes

This profiler is applicable to those organic chemicals that have presence of at least one of the 137 protein binding potency alerts specified within the profiler. The presence of alerts is not bounded with parametric ranges; it is based on structural boundaries only.

 

(6) Protein binding alerts for skin sensitization according to GHS:

The profiler is based on recently developed OASIS TIMES model for predicting skin sensitization according to GHS criteria. The protein binding alerts are extracted from 517 chemicals used as a training set for the model. In the current version of the profiler are available 131 alerts, classifying chemicals into GHS categories 1A and 1B.  The borders of Category 1B are slightly modified in the model and respectively in the profiler. As a result the classification thresholds are the following:

 - Category 1A – EC3 (LLNA) ≤ 2%; NOEL (HRIPT)≤ 500 μg/cm²

 - Category 1B – 2%;  < EC3 (LLNA) < 50%; 500 μg/cm² < NOEL (HRIPT) < 12 500 μg/cm²

 -  No alert found – EC3 (LLNA) ≥ 50%; Negative (LLNA); NOEL (HRIPT) > 12 500 μg/cm²

Each alert is associated with reaction mechanistic domain, e.g. Schiff base formation, Michael addition, Acylation, etc. and it is specified in the help file supporting the mechanism of interaction of the alerting group with skin proteins.

The profiler consists of structural and parametric (log KOW) requirements.

Relevance/Applicability to particular chemical classes

This profiler is applicable to those organic chemicals that have presence of at least one of the 135 protein binding alerts specified within the profiler. The presence of some protein binding alerts is conbined with specific logKow ranges, i.e. the profiler contains structural and parametric boundaries. The absence of a protein binding alert should not be taken as an absence of toxicity.

 

(7) Protein binding alerts for skin sensitization by OASIS:

The scope of this profiler is to investigate the presence of alerts within the target molecules responsible for interaction with proteins and especially with skin proteins. This profiler accounts for incapability of some chemicals having an alert to interact with skin due to electronic and steric factors. This is explicitly defined by inhibition masks associated with some alerts. The list of 110 structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

Relevance/Applicability to endpoint(s)

The profiler is intended to be used for the assessment of protein binding interaction of chemicals and especially interaction with skin proteins. The profiler has been developed based on mechanistic knowledge for skin sensitisation of dataset of 881 chemicals tested by Local Lymph Node Assay (LLNA) or Guinea Pig Maximization Test (GPMT). A list of 107 structural alerts has been derived, based on the mechanistic knowledge of training set chemicals. The list of 107 structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

Relevance/Applicability to particular chemical classes

This profiler is applicable to those organic chemicals that have presence of at least one of the 107 protein binding alerts specified within the profiler. The presence of protein binding alerts is not bounded with parametric ranges; it is based on structural boundaries only. The absence of a structural alert should not be taken as an absence of toxicity.

(8) Protein Binding Potency h-CLAT:

h-CALT: This profile is built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization. It is developed on the base of data derived from the human cell line activation (h-CLAT) assay. The h-CLAT is an in vitro method proposed to address the third key event (dendritic cell activation) of the skins sensitisation AOP by quantifying changes in the expression of cell surface markers associated with the process of activation of DC (i.e. CD86 and CD54), in the human leukemia cell line THP-1, following exposure to sensitizers (8). The measured expression levels of CD86 and CD54 cell surface markers are then used for supporting the discrimination between skin sensitisers and non-sensitisers.

The profile contains 30 structural alerts extracted from 223 chemicals with positive/negative data values. In cases of chemicals with available more than one value, the positive value has been used in the boundaries development.

The profiling results outcome assigns a target to the corresponding potency category based on matched structural criteria.

Relevance/Applicability to particular chemical classes

This profiler is applicable to chemicals containing at least one alert listed in the profiler. Absence of alerts in the molecular structure may be associated to inability of chemicals to interact with proteins, or may be due to a lack of mechanistic knowledge. Therefore, the ‘No structural alert’ flag is not equivalent to a negative prediction.

(9) Kerationocyte gene expression:

This profile is built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization. It is developed on the base of data derived from the KeratinoSens assay, which examined the potential for chemicals to induce the expression of a luciferase reporter gene under control of a single copy of the ARE element of the human AKR1C2 gene stably inserted into immortalized human keratinocytes. Relevance to skin sensitization is inferred from the relationship of Keap1-Nrf2-ARE regulatory pathway and its detection of electrophilic chemicals to sensitization. All included categories are defined by analisysn experimental data for EC1.5 values (the concentration eliciting a 1.5-fold increase in luciferase induction).  No gene induction is observed when EC1.5 is >2000.

The profile contains 18 structural alerts extracted from about 300 chemicals comprised within EC1.5 data. The set of 18 structural alerts are separated into four categories: very high gene expression, high gene expression, moderate gene expression and low gene expression.  Classification of categories depends on the EC1.5 values and is as follows: chemicals having very high gene expression (EC1.5< = 15 uM); high gene expression (EC1.5 = 15 – 50 uM); moderate gene expression (EC1.5 = 50 – 100 uM); low gene expression (EC1.5 = 100 – 1999 uM).

The profiling results outcome assigns a target to the corresponding potency category based on matched structural criteria.

Relevance/Applicability to particular chemical classes

This profiler is applicable to those organic chemicals that have presence of at least one of the 21 structural alerts specified within the profiler. The presence of alerts is not bounded with parametric ranges; it is based on structural boundaries only. The absence of a structural alert should not be taken as an absence of potency to interact with proteins and especially with keratinocytes.

(10) Respiratory sensitization:

This profiler is intended to be used for the assessment of respiratory sensitisation potential of low molecular weight chemicals. The profiler has been developed from mechanistic knowledge of the elicitation phase of respiratory sensitisation, thus identifies chemicals able to covalently bind to proteins in the lung. This mechanistic hypothesis makes the profiler suitable for identifying chemicals capable of inducing respiratory sensitisation via both the skin and lung (as the chemistry (for a given structural alert) must be the same in both the induction and elicitation phases of sensitisation.

Relevance/Applicability to particular chemical classes

This profiler is applicable only to organic chemicals that have a molecular weight less than 1000 g/mol. It is applicable only to the chemical classes for which it contains structural alerts; the absence of a structural alert should not be taken as an absence of toxicity.

(11) Skin sensitisation for DASS:

The customized profiler “Skin sensitization for DASS” is developed for Defined approaches (DA) purposes and it is part of the “Skin sensitization for defined approaches” automated workflow. The profiler combines application of the endpoint specific “Protein binding alerts for skin sensitization by OASIS” profiler with the Autoxidation and Skin metabolism simulators. Thus, it can identify the presence or absence of protein binding alerts in the parent chemical and predicted metabolites.

The “Skin sensitization for DASS” profiler is automatically applied on the structures, for which the AW falls to make a prediction.

1. If protein binding alert (PBA) is identified in the parent chemical and/or in the predicted metabolites, then “Positive” result appears for the target chemical. This result is interpreted as positive prediction in the context of DA. Alerts are justified mechanistically. Documentation for each of the alert is available in the Toolbox. However, no report can be generated as such under the Report module of the Toolbox.


2. If no PBA is identified in the parent chemical or in the predicted metabolites, then “Negative” result appears for the target chemical. This result could be interpreted as negative prediction in the context of DA. No report can be generated for these.


3. In case the target substance is a mixture, then more than one result is obtained (a result for each of the components). If results are same for all mixture components, then it is assigned to the mixture. If positive and negative results are profiled simultaneously (i.e. “Protein binding alert found” and “No alert found” result), then the worst-case scenario is taken and the positive result is assigned to the mixture. Documentation for each alert is available in the Toolbox. However, no report can be generated as such under the Report module of the Toolbox.

 

 

 

 

 

The above depicted table indicates that there are no structural or mechanistic alerts for skin sensitization for Estradiol with the OECD QSAR Toolbox Profilers. Although, these profiling results cannot be considered as negative results, they substantiate that Estradiol is unlikely a skin senisitizer. Based on these profilers, among others, structurally similar structures were requested using the OECD QSAR Toolbox. With a minimum of 70% structural similarity as criterion for exclusion (for substances with < 70% similarity) a metabolite of Estradiol, Estrone was detected as substance with experimental data on skin sensitization. Estrone was found to be a non-sensitizer. This result further confirm the assumption that Estradiol is not a skin sensitizer.

 

In a QSAR prediction using DEREK Nexus v6.1 the potential of Estradiol to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features) <inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.

 

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Furthermore, the query structure does not contain an unclassified feature and is consequently not predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test.

Based on these results Estradiol is considered non-mutagenic as predicted by DEREK Nexus.

 

In summary, in a Weight of Evidence Approach, all available data clearly showed that estradiol is neither skin sensitizing in animals, in humans nor in an in silico approach, thus, based on these results Estradiol is not classified according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

 

 

 

 

References:

 

Alberti et al.: Pharmaceutical development and clinical effectiveness of a novel gel technology for transdermal delivery. Expert Opin Drug Deliv. 2, 935-950, 2005

 

Archer et al.: A randomized comparison of continuous combined transdermal delivery of estradiol-norethindrone acetate and estradiol alone for menopause. CombiPatch Study Group. Obstet Gynecol. 4, 498-503,1999

 

Chetkowski et al.: Biologic effects of transdermal estradiol. N Engl J Med. 314, 1615 – 1620, 1986

 

Nath & Sitruk-Ware: Parenteral administration of progestins forhormonal replacement therapy. Europ. J. Contraception Reprod. Health 14, 88-96, 2009

 

Rozenberg et al.: Comparison of continuous and sequential transdermal progestogen with sequential oral progestogen in postmenopausal women using continuous transdermal estrogen: vasomotor symptoms, bleeding patterns, and serum lipids. Int J Fertil Womens Med. 42 Suppl 2:376-87, 1997

 

Stropp, G., 2017, Assessment of the skin sensitization potential of Estradiol

 

Utian: Transdermal estradiol overall safety profile. Am J Obstet Gynecol. 156, 1335 - 1338, 1987

 

Utian et al.: Efficacy and safety of low, standard, and high dosages of an estradiol transdermal system (Esclim) compared with placebo on vasomotor symptoms in highly symptomatic menopausal patients. The Esclim Study Group. Am J Obstet Gynecol. 181, 71-79, 1999

 

Wiseman & McTavish: Transdermal estradiol/norethistherone. A review of its pharmacological properties and clinical use in the postmenopausal women. Drugs Aging 4, 238-256, 1994

 

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on animal data for surrogate and in the light of the clinical experience with estradiol no classification is required according to Regulation (EC) No. 1272/2008 (CLP), Annex I.