Oestr-5(10)-ene-3,17-dione

Endpoint summary

Description of key information

The test substance has no skin senitizing potential. (Leidenfrost 2016)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov. 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Guideline adopted 22 July 2010; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). The IMDS was validated and published with scientific justification in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

Deviations:

yes

Remarks:

modifications: 1. non-radioactive alternative, measuring lymph node cell counts; 2. in addition, measurement of ear swelling and ear weight to discriminate the irritating potential from the sensitizing potential of the test substance

Principles of method if other than guideline:

This study is performed according to OECD TG 429. As stated in OECD TG 429 besides the classical radioactive method ‘other endpoints for assessment of the number of proliferating cells may be employed’ as so-called ‘me-too’ tests, if the required performance standards are fulfilled, they are ‘based on similar scientific principles and measure or predict the same biological or toxic effect’ and they are validated.
Here, an alternative method is used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes (IMDS LLNA; Integrated Model for the Differentiation of Skin Reactions). In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitizing properties. Information on validation of the IMDS LLNA and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
In the IMDS LLNA stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify skin sensitization) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
- Vohr, H.-W., Blümel, J., Blotz, A., Homey, B. and Ahr, H.J. An intra-laboratory validation of IMDS: Discrimination between (Photo) Allergic and (Photo) Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509 (2000).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 28-34 g
- Housing: 1 animal/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 40-70 %
- Air changes (per hr): about 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

0, 2, 10, 30 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control (30 % alpha hexyl cinnamic aldehyde) in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2, d3). The volume administered was 25 µl/ear/day. Based on our experiences with the test system and the expected solubility of the test item the following concentrations were used: 0 % (vehicle control), 2 %, 10 % and 30 %. A preparation of a formulation >30 % was not possible. One day after the last application (day 4) the animals were anaesthetized by inhalation of carbon dioxide and sacrificed. The lymphatic organs (the auricular lymph nodes) were removed and transferred into physiological saline (PBS).

INVESTIGATIONS:
- weight of the lymph nodes (given as stimulation index compared to vehicle treated control group)
- cell counts of lymph nodes (given as stimulation index compared to vehicle treated control group; positive if greater or equal as 1.4 stimulation index )
Stimulation indices were calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (positive, if 2 x 10-2 mm increase; 10 % of the control value)
- ear weight (8 mm diameter piece of the right and left ear of each animal by using a punch)

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The individual values from actively treated groups were compared with those from the control
group. A pre-testing was carried out by a Cochran test. Furthermore, depending on the
statistical result, a Bonferroni-Holm test (Mann-Whitney test included) or a Dunnett test
significance test was conducted (significance levels of 5 %; two-tailed).
In this method of statistical processing of measurements a large number of comparisons are
made, and as a result of the multiple tests the overall probability of error is considerably
greater than the p values suggest (increased number of false-positive results). On the other
hand, the known methods of adjusting p values lead to an excessive increase in the number of
false negatives. In view of these problems the biological and toxicological relevance is also
taken into consideration in the evaluation of statistical significance.
For this reason, in the case of indices only the standard deviations between groups and
difference analysis of the mean values were used for the final evaluation of the biological
relevance.

Positive control results:

Positive control - sensitizing potential: After treatment with Alpha Hexyl Cinnamic Aldehyde the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals. The positive level
which is 1.4 for cell count indices, clearly was exceeded.
Positive control - irritating potential: The positive level of ear swelling, which is 2x10-2 mm increase, i.e. about 10 % of the control values, was exceeded in the positive control group. This increase is of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was also detected.

Key result

Parameter:

SI

Value:

1.94

Variability:

37.11

Test group / Remarks:

30% positive control

Key result

Parameter:

SI

Value:

1.33

Variability:

52.79

Test group / Remarks:

30% test group

Key result

Parameter:

SI

Value:

1.01

Variability:

25.16

Test group / Remarks:

10% test group

Key result

Parameter:

SI

Value:

1.12

Variability:

28.75

Test group / Remarks:

2% test group

Key result

Parameter:

SI

Value:

1

Variability:

29.31

Test group / Remarks:

Vehicle

Cellular proliferation data / Observations:

The NMRI mice did not show increases in stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item. The positive level indicating sensitizing potential, which is 1.4 for the cell count index, was never reached or exceeded in any dose group (see Table 1). The positive level of ear swelling indicating irritating potential, which is 2 x 10-2 mm increase, i.e. about 10 % of the control values, was not reached or exceeded in any dose group (See Table 2).

Table 1: Weight index, Cell count index

Groups	Weight index(index of mean ± SD in %)	Cell count index (index of mean ± SD in %)
Vehicle	1.00 ± 25.49	1.00 ± 29.31
2 % test item	1.19 ± 24.33	1.12 ± 28.75
10 % test item	1.12 ± 16.00	1.01 ± 25.16
30 % test item	1.61 ± 44.76	1.33 ± 52.79
30 % positive control	1.49* ± 31.06	1.94* ± 37.11

* statistically significant increase (p < 0.05)

Table 2: Ear swelling

Groups	Day 1(Ear swelling in mm x10-2)(mean ± SD in %)	Day 4(Ear swelling in mm x10-2)(mean ± SD in %)	Index Day 4
Vehicle	17.33 ± 2.84	17.75 ± 2.55	1.00
2 % test item	17.33 ± 2.84	17.58 ± 2.93	0.99
10 % test item	17.33 ± 2.84	17.50 ± 3.85	0.99
30 % test item	17.17 ± 3.36	17.92 ± 4.43	1.01
30 % positive control	17.83 ± 3.24	26.08* ± 13.53	1.47

* statistically significant increase (p < 0.05)

Table 3: Ear weight

Groups	Day 4(ear weight in mg)(mean ± SD in %)	Index Day 4
Vehicle	12.97 ± 4.27	1.00
2 % test item	12.48 ± 3.40	0.96
10 % test item	12.00 ± 3.41	0.93
30 % test item	12.10 ± 4.47	0.93
30 % positive control	20.18* ± 9.51	1.56

* statistically significant increase (p < 0.05)

Only after treatment with the positive control the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals (table 1). The positive level, which is 1.4 for cell count indices, clearly was exceeded.

In addition, ear swelling and the ear weight were only increased after treatment with the positive control (table 2, table 3).

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was investigated in the modified local lymph node assay (LLNA-IMDS) on female NMRI mice according to OECD 429. Concentrations of 0 (vehicle control), 2, 10 or 30 % formulated in dimethylformamide were tested. Validity of the assay was demonstrated by the positive results obtained with positive control compound alpha hexyl cinnamic aldehyde.

This study did neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test item.

Taken together, no antigen specific activation of the cells of the immune system via dermal route was determined after application of up to and including 30 % test item by the method used. Therefore, the concentration of 30 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.

Executive summary:

In a dermal sensitization study according to OECD TG 429 (24 April 2002) with Delta-5-Norandrostendion in Dimethylformamide, young adult female NMRI Mice (6/dose) were tested in the LLNA (OECD TG 429). Up to 30% of the test item was applied. Hexyl cinnamic aldehyde (CAS No 101-86-0) was used as positive control substance. Only after treatment with the positive control, alpha hexyl cinnamic aldehyde, the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals.

This study did neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test substance. In this study, the test item is not a dermal sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a dermal sensitization study according to OECD TG 429 (24 April 2002) with Delta-5-Norandrostendion in Dimethylformamide, young adult female NMRI Mice (6/dose) were tested in the LLNA (OECD TG 429). Up to 30% of the test item was applied. Hexyl cinnamic aldehyde (CAS No 101-86-0) was used as positive control substance. Only after treatment with the positive control, alpha hexyl cinnamic aldehyde, the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals.

This study did neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test substance. In this study, the test item is not a dermal sensitizer.

 

In a dermal sensitization study similar to OECD test guideline 406 (Buehler test) with 5-Delta-Norandrostendione (0.200 g a.i.in 0.2 mL induction/challenge solution = 50%) in 80% ethanol (induction) and acetone (challenge), Dunkin-Harley guinea pigs (Liaoning Changsheng Biotechnology Co., Ltd.), five weeks of age and between 337.81 and 388.90 g were tested using the method of Buehler2-mercaptobenzothiazole was used as the positive control substance to carry out the reliability check using the test methodology for this test. 0.2 mL 50% 2-mercaptobenzothiazole solution (medium: 80% ethanol) was used for the induction exposure, 0.2 mL 2.5% 2-mercaptobenzothiazole solution (medium: acetone) was used for the challenge exposure. The skin sensitisation rate was 30%, confirming that the methodology used in this test and animal sensitivity were valid.

On day 28 the animals were topically challenged with 50% test sample solution for six hours. The 24h reading in the test group revealed that one animal showed a discrete erythema which had disappeared at the 48h reading. No such erythema was found in animals treated in the same manner as the test groups but without test item. The erythema detected in one animal of the test group was also found in 15 animals of the same group during induction procedure, whereas the control animals showed no effects. According to the classification criteria of the guideline the skin sensitisation rate for the animals in the test group was 5%.

In this study, 5-Delta-Norandrostendione is not a dermal sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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