4-hydroxybutan-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Feb 2015 - 04 Mar 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

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:

4-hydroxybutan-2-one

EC Number:

209-693-6

EC Name:

4-hydroxybutan-2-one

Cas Number:

590-90-9

Molecular formula:

C4H8O2

IUPAC Name:

4-hydroxybutan-2-one

Details on test material:

- Name of test material (as cited in study report): 4-Hydroxybutan-2-one
- Test Item No.: 14/0671-1
- Physical state: Colorless , clear liquid
- Analytical purity: 96.2%; 96.1% corrected to the water content
- Homogenicity: The test item appeared to be homogeneous
- Lot/batch No.: 10781727
- Expiration date of the lot/batch: 04 November 2016
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: pre-test and main study 10 – 11 weeks
- Housing: group housing Makrolon Type II (pre-test) Makrolon Type III cages (main study), with w ire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Relative Humidity (%): approx. 45 - 65%
- Photoperiod (hrs dark / hrs light):artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

25, 50 and 100%

No. of animals per dose:

2 females for the pre-test ; 5 females per group (main-study)

Details on study design:

RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used, was 100% of the undiluted test item. Test item solutions at different concentrations were prepared using DMF as vehicle. Vortexing was used to formulate the test item.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in
two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily
each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were
recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin.
Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a
micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals showed neither any signs of local skin irritation nor systemic toxicity.
Thus, the test item in the main study was assayed at 25, 50, and 100%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Furthermore, an index was calculated for the lymph node weight and –cell count as well as for the ear weight by dividing the mean values of the test
item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was
reported for a positive response and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1.
However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be
implicitly adopted.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and DMF was added. The different test item concentrations were prepared
individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 25 nd 50% in DMF as well as 100% (undiluted test item) . The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear
once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone
(control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.5 µCi of ³H-methyl thymidine (equivalent to
81.8 µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node
cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations validated statistical
program R Script Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program R Decision Tree.
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control
experiment was performed using CBA/CaOlaHsd mice in October 2014.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices (S.I.) of 1.88, 4.24, and 4.94 were determined with the test item at concentrations of 25 and 50% (w/w) in DMF, as well as with 100% (undiluted test item), respectively. A clear dose response was observed and an EC3 value of 36.9 % was derived.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase in lymph node cell counts was observed in the mid and high dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was reached in the low dose group and exceeded in the mid and high dose group (indices of 1.55, 1.93 and 2.05). A statistically significant or biologically relevant increase in lymph node weights was not observed in any treated group in comparison to the vehicle control group.

Any other information on results incl. tables

Calculation of Stimulation Indices per Dose Group:

Test item concentration	Group Calculation	SD	S.I.
	Mean DPM per animal (2 lymph nodes)^a)
Vehicle Control (DMF)	751.3	357.0	1.00
25% 4-Hydroxybutan-2-one	1409.6	539.8	1.88
50% 4 -Hydroxybutan-2-one	3185.8	1056.4	4.24*
100% 4-Hydroxybutan-2-one	3711.0	1675.5	4.94*

a)  Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within

a group by the number of animals in that group (5 animals, except for the vehicle group with only 4 animals, since one animal was

excluded from the calculations)

*   statistically significant (p<0.05).

Calculation of the EC3 value:

	Test item concentration %	S.I.
Test Group 2	25 (a)	1.88 (b)
Test Group 3	50 (c)	4.24 (d)

EC3=(a-c)[(3 -d)/(b-d)]+c=36.9%(w/w)

Mortality

No deaths occurred during the study period.

Clinical Signs

Neither signs of systemic toxicity nor local skin effects were observed during the study period.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Ear Weights

The measured ear weights of all animals treated were recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test item 4-Hydroxybutan-2-one was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

In this study the test item 4-Hydroxybutan-2-one was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solutions at different concentrations were prepared in the vehicle DMF (dimethylformamide).

The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations.

For this purpose a local lymph node assay was performed using test item concentrations of 25 and 50% (w/w) as well as 100% (undiluted test item). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment).

The animals showed neither any signs of systemic toxicity nor local skin effects during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation (see Ref. 9). None of the indices determined for the test item treated groups reached or exceeded this threshold.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 1.88, 4.24, and 4.94 were determined with the test item at concentrations of 25 and 50% in DMF, as well as with 100% (undiluted test item), respectively. A clear dose response was observed.

An outlier was not identified in any group.

A statistically significant and biologically relevant increase in DPM value and also in lymph node cell counts was observed in the mid and high dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was reached in the low dose group and exceeded in the mid and high dose group (indices of 1.55, 1.93 and 2.05).

A statistically significant or biologically relevant increase in lymph node weights was not observed in any treated group in comparison to the vehicle control group.

The test item 4-Hydroxybutan-2-one was found to be a skin sensitizer and an EC3 value of 36.9 % was derived.