γ-butyrolactone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

Study in animals performed before Annex VII and VIII amendments.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jan 2010 thru 5 Feb 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

included in report

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

γ-butyrolactone

EC Number:

202-509-5

EC Name:

γ-butyrolactone

Cas Number:

96-48-0

Molecular formula:

C4H6O2

IUPAC Name:

dihydrofuran-2(3H)-one

Details on test material:

- Name of test material (as cited in study report): y-Butyrolactone (CASRN: 96-48-0)
- Analytical purity: 99.86 area-%
- Homogeneity: Homogeneous by visual inspection
- Test Substance Number: 05/0603-2
- Lot/batch No.: Tank 62 Production date 9.12.2009
- Expiration date of the lot/batch: stable over the study period

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.1 g – 20.9 g
- Housing: Makrolon cage, type II
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 30 – 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)

IN-LIFE DATES: From: 20 Jan 2010 To: 01 Feb 2010

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

Undiluted, 60%, and 30%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Soluble in vehicle (MEK)
- Irritation: No
- Lymph node response: Slight increase in Lymph node weights after application of undiluted substance

MAIN STUDY

The skin sensitizing potential of y-butyrolactone was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/J mice each were treated with the undiluted test substance and with 60% and 30% w/w preparations of the test substance in MEK (methyl ethyl ketone) or with the vehicle alone. A concurrent positive control group with 5 female CBA/J mice was treated with 10% Alpha-Hexylcinnamaldehyde, techn. 85% (HCA) in MEK.

The study used 3 test groups, 1 control group and 1 positive control group. Each test animal was treated with 25 µL per ear of the appropriate test-substance preparation, applied to the dorsal surface of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone. The animals of the positive control group were treated with 25 µL of a 10% preparation of Alpha-Hexylcinnamaldehyde in MEK.

Three days after the last application the mice were injected into a tail vein with 20 µCi of 3H-thymidine in 250 µL of sterile saline. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

The increase in 3H-thymidine incorporation by a factor of ≥ 3 (SI ≥ 3) as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. Cell count, 3H-thymidine incorporation, lymph node weight and ear weight results were analyzed by the WILCOXON test.

Results and discussion

Positive control results:

Valid. The 10% HCA preparation in MEK induced a biologically relevant and statistically significant response in 3H-thymidine incorporation (SI ≥ 3.0) as well as statistically significant increased lymph node cell counts and lymph node weights.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.06

Test group / Remarks:

30%

Results 2

Key result

Parameter:

SI

Value:

0.96

Test group / Remarks:

60%

Results 3

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

100%

Any other information on results incl. tables

Test Group	Treatment	Cell Count Stimulation Index	^3H-thymidine incorporation Stimulation Index	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
1	vehicle MEK	1.00	1.00	1.00	1.00
2	30% in MEK	1.08	1.18	1.06	1.02
3	60% in MEK	0.91	0.97	0.96	0.99
4	undiluted	0.87	1.28	1.01	1.01
5	positive control	1.92##	5.20##	1.74##	1.14##

The statistical evaluations were performed using the WILCOXON-test (# for p<=0.05, ## for p<=0.01)

 

Test Group	Treatment	Cell Counts [Counts/Lymph Node Pair]
		Mean	S.D.	Stimulation Index*
1	vehicle MEK	7,005,600	933,483	1.00
2	30% in MEK	7,561,200	835,901	1.08
3	60% in MEK	6,394,800	345,753	0.91
4	undiluted	6,127,800	1,563,684	0.87
5	positive control	13,473,600	1,520,065	1.92 ##

 

Test Group	Treatment	Lymph Node Weight [mg/Lymph Node Pair]
		Mean	S.D.	Stimulation Index*
1	vehicle MEK	4.3	0.4	1.00
2	30% in MEK	4.5	0.6	1.06
3	60% in MEK	4.1	0.3	0.96
4	undiluted	4.3	0.5	1.01
5	positive control	7.4	0.7	1.74 ##

Test Group	Treatment	Ear Weight [mg/animal]
		Mean	S.D.	Stimulation Index*
1	vehicle MEK	29.2	1.8	1.00
2	30% in MEK	29.8	1.0	1.02
3	60% in MEK	29.0	1.5	0.99
4	undiluted	29.4	1.4	1.01
5	positive control	33.3	1.8	1.14 ##

*test group x / test group 1 (vehicle control)

# = statistically significant value p<=0.05

## = statistically significant value p<=0.01

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

y-Butyrolactone does not show a skin sensitizing effect in the Murine Local Lymph Node Assay.