19-Norpregn-5(10)-ene-21-nitrile, 17-hydroxy-3-oxo-, (17.alpha.)-

Endpoint summary

Description of key information

Skin irritation (rat): not irritating [Kurth 2009a] - Skin irritation (in vitro): not irritating [Meyer and Steinhoff 2006]
Eye irritation (rabbit): not irritating [Kurth 2009b]

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July to Nov 2006

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

24 April 2002

Deviations:

yes

Remarks:

- exposure time 24 hrs instead of 4 hrs, few details in test description and reporting

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

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Weight at study initiation: males: 283 – 290 g; females: 208 – 218 g
- Housing: individually in conventional cages
- Diet (e.g. ad libitum): pell. Ssniff® R / M - H, ad libitum, 24 hours per day
- Water (e.g. ad libitum): filtrated tap water, ad libitum, 24 hours per day
- Acclimation period: ≥ 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 56-59
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

physiological saline

Controls:

other: the untreated skin served as control

Amount / concentration applied:

566-580 mg / male animal or 417-436 mg / female animal (= 2000 mg/kg bw)

Duration of treatment / exposure:

24 hours

Observation period:

21 days

Number of animals:

3 males and 3 females

Details on study design:

- Duration of observation period following administration: 21days
- Frequency of observations and weighing: All alterations of the baseline condition of the animals were recorded. All animals were checked four times on administration day and once daily on the following days up to day 21 of the test. All alterations of the skin at the administration sites of all animals were recorded according to the scoring system recommended by the EU. The application sites were evaluated 1, 24, 48 and 72 h after removal of the patches. Body weight was determined at the start (day 1), on days 8 and 15 and at the end of the study (day 21).
- Necropsy of survivors performed: yes

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no irritation observed

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No irritation observed

No compound-related findings were observed at the dose of 2000 mg/kg. The mean values of findings relevant for classification (reddening, scab formation and swelling of the skin) at the time-points 24, 48 and 72 hours after the end of administration were 0 for swelling, reddening and scab formation.

Conclusions:

A single dermal administration of the test substance for 24 hours to male and female rats at a dose of 2000 mg/kg (566-580 mg/male or 417-436 mg/ female) was tolerated without any local irritations. The mean values of findings relevant for classification (reddening, scab formation and swelling of the skin) at the time-points 24, 48 and 72 hours after the end of administration were 0 for swelling, reddening and scab formation.

Executive summary:

In a primary dermal irritation study similar to OECD TG 404 (adopted 24 April 2002), young adult Wistar rats (3/sex) were dermally exposed to 566-580 mg (males) and 417-436 mg (females) of 5-Dehydrocyanomethylketon in physiological saline for 24 hours). Animals then were observed for 21 days.  Irritation was scored by the method described in the guideline.

The mean values of findings relevant for classification (reddening, scab formation and swelling of the skin) at the time-points 24, 48 and 72 hours after the end of administration were 0 for swelling, reddening and scab formation.. In this study, 5-Dehydrocyanomethylketon is not a dermal irritant.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May to June 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

The test substance was investigated regarding its potential irritating properties using reconstituted epidermis models. The test uses topical exposure of the neat test chemical to the MatTek human reconstituted epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), present in mitochondria of viable cells, into a blue formazan salt that is quantitatively determined after extraction from tissues. The reduction of viability of tissues exposed to chemicals in comparison to neagtive controls (treated with water) is used to predict skin irritation potential. In addition, the release of IL-1 alpha was determined after approximately 42 hours of incubation to investigate another endpoint for the classification of skin irritation potential.

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Vehicle:

other: aqua dest.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: RHE/S/17: Reconstituted Human Epidermis small, age day 17,
SkinEthic Laboratories, Nice, France
- Tissue batch number(s): 05 022A 0501
- Date of initiation of testing: Passage: 2nd strain n° PK2 GHI 25

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: n/a

NUMBER OF REPLICATE TISSUES: triplicate


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater than or equal to 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 439: Study was conducted prior to adoption of OECD guideline 439.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 mg

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min

Value:

75.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, tissue viability is meeting the acceptance criterion if the mean absorption of the three tissues is ≥ 1.
- Acceptance criteria met for positive control: Yes, the mean relative tissue viability after a 15 minutes exposure and an incubation period of approximately 42 hour is ≤ 20 % of the negative control.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation calculated from individual percentual tissue viabilities of the 3 identically treated replicates is < 30.
- Range of historical values if different from the ones specified in the test guideline: n/a

Table 1: Prediction of irritation potential from MTT results

 

Compound	Cell viability [%]	Classification
Test item	75.5	non-irritant
Positive control	1.4	irritant
Negative control	100.0	non-irritant

 

The test item was characterised by no significant impact on cell viability after a 15 min. exposure period.

 

Standard deviation of the 3 tissue replicates

Test substance	SD [%]
Test item	22.64
Negative control	3.19
Positive control	5.84

 

Table 2: Interleukin-1 alpha analysis

 

Compound	IL-1 alpha [pg/mL medium] ± SD	Fold-increase of IL-1 alpha release compared to the negative control
Test item	32.5 ± 17.08	4.9
Positive control	745.3 ± 16.97	112.6
Negative control	6.6 ± 1.05	1.0

 

 

Conclusions:

The test item was investigated regarding its potential skin irritating properties using EpiDerm reconstituted epidermis models. After 15 minutes of exposure to the neat substance and an additional incubation period of approximately 42 hours cell viability of the skin models treated with the test item was around 75 % of the cell viability of the negative control. Therefore, the test item can be classified as non-irritating compound. In addition, the very low concentration of the proinflammatory IL-1 alpha released into the assay medium after approximately 42 hours of substance exposure supports this finding.

Executive summary:

In a dermal irritation study performed in accordance with but prior to the initial adoption of OECD Guideline 439 (In Vitro Skin Irritation), 5-Dehydromethylketon (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. Each approximately 20 mg of the test item were applied to the tissues.

After 15 minutes exposure, the tissue constructs were incubated for 42 h postincubation period. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 75.5%. Since the mean relative tissue viability for the test substance was above 50%, the test item is identified to be not irritating.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2007

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:

24 April 2002

Deviations:

yes

Remarks:

- 4 instead of 3 animals

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kisslegg, Germany
- Weight at study initiation: males: 4.0 - 4.3 kg; females: 4.3 - 5.2 kg
- Housing: individually in conventional scanbur cages
- Diet (e.g. ad libitum): pell. Ssniff ® K - H ad libitum
- Water (e.g. ad libitum): filtrated tap water ad libitum
- Acclimation period: ≥ 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 50-54
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: the left eye, which remained untreated, served as control.

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

test substance remained in the eye (eye was not rinsed)

Observation period (in vivo):

4 days

Number of animals or in vitro replicates:

2 males and 2 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Substance was not removed
SCORING SYSTEM: Evaluation of the local tolerance on the conjunctiva of the rabbits was performed according to the score system recommended for the EU (Annex to EEC guideline 92/69/EWG, dated 31 Jul 1992, for the 17th adaptation of the EEC guideline 67/548/EWG, B 5. Acute Toxicity (Eye irritation).Off J EEC, L383A, Luxembourg, 29 Dec 1992.).

TOOL USED TO ASSESS SCORE: Not reported.

Irritation parameter:

conjunctivae score

Remarks:

swelling

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

swelling

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

swelling

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

swelling

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

reddening

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

reddening

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

reddening

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Remarks:

reddening

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Max. score:

4

Remarks on result:

not measured/tested

Remarks:

Chemosis score refers to the swelling of eye lids and nictating membranes. In this test only the swelling and reddening of the conjunctiva was reported

Table 1: Results of the study

 

		Irritant effects (score)
Animal no. (sex)	Irritation parameter	24 h	48 h	72 h	Mean scores
1 (M)	Cornea	0	0	0	0.0
	Iris	0	0	0	0.0
	Conjunctiva (reddening)	1	1	0	0.67
	Conjunctiva (swelling)	0	0	0	0.0
2 (M)	Cornea	0	0	0	0.0
	Iris	0	0	0	0.0
	Conjunctiva (reddening)	0	0	0	0.0
	Conjunctiva (swelling)	0	0	0	0.0
3 (F)	Cornea	0	0	0	0.0
	Iris	0	0	0	0.0
	Conjunctiva (reddening)	1	0	0	0.33
	Conjunctiva (swelling)	1	0	0	0.33
4 (F)	Cornea	0	0	0	0.0
	Iris	0	0	0	0.0
	Conjunctiva (reddening)	1	1	0	0.67
	Conjunctiva (swelling)	1	0	0	0.33

 

The control eyes were without findings.

 

Local findings after single administration of the test item into the conjunctival sac of the rabbit eye were slight to severe reddening and slight to moderate swelling of the conjunctivae in all animals on administration day. On day 2 slight reddening and/or swelling of the conjunctivae occurred in 3 out of 4 animals. All animals were without findings 72 hours after instillation of the test substance.

Conclusions:

In a local tolerance test on the rabbit eye according to OECD TG 405 a single application of the test substance into the conjunctival sac provoked slight to moderate irritation of the conjunctivae from administration day on up to day 2 and were fully reversible 72 hours after administration. The mean values of findings relevant for classification at the time-points 24, 48 and 72 hours after application were 0 for the parameters cornea and iris, 0.17 for conjunctival swelling and 0.42 for conjunctival reddening . According to EU classification criteria ZK 37877 need not be labelled as an eye irritant.

Executive summary:

In a primary eye irritation study according to OECD TG 405 (adopted 24 April 2002), 0.1 mL corresponding to 56.0 – 58.2 mg of 5-Dehydromethylketon (100% a.i.) as undiluted test material was instilled into the conjunctival sac of right eye of (2/sex) New Zealand White rabbits (Charles River, Kisslegg, Germany, males: 4.0-4.3 kg and females 4.3-5.2 kg). Animals were then observed for 4 days. Irritation was scored by the method described in the guideline.

Local findings after single administration of the test item into the conjunctival sac of the rabbit eye were slight to severe reddening and slight to moderate swelling of the conjunctivae in all animals on administration day. On day 2 slight reddening and/or swelling of the conjunctivae occurred in 3 out of 4 animals. All animals were without findings 72 hours after instillation of the test substance. In this study, the test item is not an eye irritant.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a primary dermal irritation study similar to OECD TG 404 (adopted 24 April 2002), young adult Wistar rats (3/sex) were dermally exposed to 566-580 mg (males) and 417-436 mg (females) of 5-Dehydrocyanomethylketon in physiological saline for 24 hours). Animals then were observed for 21 days.  Irritation was scored by the method described in the guideline.

The mean values of findings relevant for classification (reddening, scab formation and swelling of the skin) at the time-points 24, 48 and 72 hours after the end of administration were 0 for swelling, reddening and scab formation.. In this study, 5-Dehydrocyanomethylketon is not a dermal irritant.

 

In a dermal irritation study performed in accordance with but prior to the initial adoption of OECD Guideline 439 (In Vitro Skin Irritation), 5-Dehydromethylketon (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. Each approximately 20 mg of the test item were applied to the tissues.

After 15 minutes exposure, the tissue constructs were incubated for 42 h postincubation period. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 75.5%. Since the mean relative tissue viability for the test substance was above 50%, the test item is identified to be not irritating.

 

In a primary eye irritation study according to OECD TG 405 (adopted 24 April 2002), 0.1 mL corresponding to 56.0 – 58.2 mg of 5-Dehydromethylketon (100% a.i.) as undiluted test material was instilled into the conjunctival sac of right eye of (2/sex) New Zealand White rabbits (Charles River, Kisslegg, Germany, males: 4.0-4.3 kg and females 4.3-5.2 kg). Animals were then observed for 4 days. Irritation was scored by the method described in the guideline.

Local findings after single administration of the test item into the conjunctival sac of the rabbit eye were slight to severe reddening and slight to moderate swelling of the conjunctivae in all animals on administration day. On day 2 slight reddening and/or swelling of the conjunctivae occurred in 3 out of 4 animals. All animals were without findings 72 hours after instillation of the test substance. In this study, the test item is not an eye irritant.

 

 

Justification for classification or non-classification

Based on the study results a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.