Hydroxyprogesterone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxyprogesterone

EC Number:

200-699-4

EC Name:

Hydroxyprogesterone

Cas Number:

68-96-2

Molecular formula:

C21H30O3

IUPAC Name:

17-hydroxypregn-4-ene-3,20-dione

Details on test material:

Purity: 99.8 %

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Strain: Crl:NMRI BR
- Females: nulliparous and non-pregnant
- Age at study initiation: 8 weeks
- Weight at study initiation: 28-33 g
- Housing: 1 animal/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

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

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 2, 10, 30 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated before each administration in dimethylsulfoxide (DMSO). The formulations were visually described as solution (2 %) or suspension (10 % and 30 %). During application the suspensions were stirred on a magnet stirrer.
The homogeneity and stability of the test item in the vehicle was analytically verified for up to 4 hours.

The test item 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 and d3). The volume administered was 25 µL/ear/day. Based on our experiences with this 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.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The lymphatic organs (the auricular lymph nodes) were then 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 (given in 0.01 mm and as index; positive, if 1.10 was exceeded)
- ear weight (given in mg / 8 mm diameter piece and as index)

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.

Results and discussion

Positive control results:

Alpha hexyl cinnamic aldehyde showed a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

cell count index

Value:

1

Variability:

+/- 36.55 %

Test group / Remarks:

Vehicle (DMSO)

Results 2

Key result

Parameter:

SI

Remarks:

cell count index

Value:

1.22

Variability:

+/- 28.09 %

Test group / Remarks:

2 % test item

Results 3

Key result

Parameter:

SI

Remarks:

cell count index

Value:

0.83

Variability:

+/- 32.84 %

Test group / Remarks:

10 % test item

Results 4

Key result

Parameter:

SI

Remarks:

cell count index

Value:

0.85

Variability:

+/- 32.17 %

Test group / Remarks:

30 % test item

Results 5

Parameter:

SI

Remarks:

cell count index

Value:

1.51

Variability:

+/- 23.08 %

Test group / Remarks:

Positive control (30 % alpha hexyl cinnamic aldehyde in DMSO)

Any other information on results incl. tables

Table 1: Summary of the LLNA results (means of 6 animals per group)

Parameter investigated	Vehicle control	Test item 2 %	Test item 10 %	Test item 30 %	Positive control
Stimulation index: weight of draining lymph nodes	1.00	1.12	0.77	0.86	1.25
Stimulation index: cell count in draining lymph nodes	1.00	1.22	0.83	0.85	1.51*
Ear swelling in 0.01 mm on day 4 (index)	18.83 (1.00)	19.75 (1.05)	19.67 (1.04)	19.50 (1.04)	21.92 * (1.11)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	14.58 (1.00)	14.02 (0.96)	13.95 (0.96)	13.86 (0.95)	17.17 * (1.22)

* statistically significant increase (p ≤ 0.05)

The 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.

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. No substance specific effects were determined for ear weights either.

The validity of the assay was demonstrated by the positive results of the positive control group (alpha hexyl cinnamic aldehyde).

The body weights of the animals were not affected by any treatment.

Applicant's summary and conclusion

Conclusions:

The results show that the test item has neither an irritating nor a sensitizing potential in mice after dermal application.

Executive summary:

The test item Hydroxyprogesteron was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 2, 10 and 30 % formulated in DMSO were tested.

The cell count indices for test item concentrations of 2, 10, and 30% were 1.22, 0.83, and 0.85, respectively. The “positive level” indicating sensitizing potential, which is 1.4 for the cell count index, was never reached or exceeded in any dose group. 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 also not reached or exceeded in any dose group.

The results show that the test item has neither an irritating nor a sensitizing potential in mice after dermal application. The validity of the assay was demonstrated by the positive results of the positive control group (alpha hexyl cinnamic aldehyde).