Butyl 3-hydroxybutyrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Butyl 3-hydroxybutyrate

EC Number:

258-658-1

EC Name:

Butyl 3-hydroxybutyrate

Cas Number:

53605-94-0

Molecular formula:

C8H16O3

IUPAC Name:

butyl 3-hydroxybutanoate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

The test substance, identified as n-butyl-3-hydroxybutyrate, Sample ID #: 30705-83-df, was received on June 6, 2013 and was further identified with PSL Reference Number 130606-5D. The test substance was stored at room temperature, in the dark. Solubility testing cinducted by PSL determined that the sample was soluble in acetone/olive oil 4:1 v/v mixture. Documentation of the methods of synthesis, fabrication, or derivation of the test substance is retained by the Sponsor.

The following information related to the characterization of the test substance was provided by the Sponsor:

Composition: n-butyl-3-hydroxybutyrate-100%, CAS #53605-94-0
Physical Description: Colorless 1 iquid
pH: 6
Solubility: The aqueous solubility is 3.9 weight%.
Stability: Test substance was. expected to be stable for the duration of testing.
Expiration Date: Stable for the duration of testing.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Housing: The animals were individually housed in plastic solid bottom cages during the dosing and resting phase of the study. After final weighing until sacrifice, animals were housed in their respective dose groups in plastic cages with bedding. Bedding in the plastic, solid bottom cages was changed at least once per week. All caging conformed to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011 ).

Animal Room Temperature and Relative Humidity Ranges: 19-22°C and 60-69%, respectively.

Animal Room Air Changes/Hour: 13. Airflow measurements are evaluated regularly and the records are kept on file at Product Safety Labs.

Photoperiod: 12-hour light/dark cycle

Acclimation Period: 7 or 14 days

Food: Harlan Teklad Global 16% Protein Rodent Diet® #2016. The diet was available ad libitum.

Water: Filtered tap water was supplied ad libitum. There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Product Safety Labs.

Cage identification: Each cage was identified with a cage card indicating at least the study number, identification, and sex of the animal.

Animal identification: Each animal was marked with a color code and given a sequential animal number assigned to study 36768, which constituted unique identification.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

No. of animals per dose:

5

Details on study design:

Three test substance concentrations and the vehicle control were used. Test substance concentrations of 25%, SO% and 100% were tested to determine the highest achievable level that avoids overt systemic toxicity and excessive local irritation. Dilutions of the test substance (25% and 50%) were prepared as w/w mixtures in AOO. Each group consisted of two mice. The ears of each mouse were scored for erythema pre-dose on Day 1 and on Days 2, 3, and 6 according to the scoring system described in Section S.E.

Twenty-five J.I.L of the appropriate concentration of the test substance or the vehicle alone was applied to the dorsum of both ears of each mouse for three consecutive days. App1ication was done using an appropriate size micropipette to accurately deliver 25 ~. The dose was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette. No treatment was made on Days 4 and 5. On Day 6, the sites for each mouse were evaluated for local reactions (erythema & edema). Animals were observed daily for signs of toxicity. The Study Director used this data in
conjunction with any pre-existing data to select the three concentrations to be tested. The test substance at 25% and 50% w/w mixtures in AOO and at 100% were selected for test.

SELECTION OF ANIMALS/DOSE LEVELS: Prior to dosing, the animals were weighed and the ears were checked for any abnormalities or clinical signs of diseases or injury. Twenty-five healthy naive female mice without pre-existing ear irritation were selected and distributed (5 mice per group) into the following test groups:

Group# Purpose Concentration
1 Vehicle Control 0%
2 Positive Control Substance 25%HCA
3 Test Substance 25%
4 Test Substance 50%
5 Test Substance 100%

Concentrations were selected based on toxicity, solubility, irritancy, and viscosity.

Positive control substance(s):

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

Statistics:

Statistical analysis was performed. Significance was judged at p <0.05. The treated groups and negative vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett's t-test for multiple comparisons. Where variances are considered significantly different by Bartlett's test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn's test) (INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using Grubbs (1969).

Results and discussion

Positive control results:

The positive control (HCA) at 25% produced a
dermal sensitization response in mice (SI = 8.34). Therefore, the LLNA test system was valid for
this study with n-butyl-3-hydroxybutyrate.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.11

Test group / Remarks:

25% test material concentration

Results 2

Key result

Parameter:

SI

Value:

1.17

Test group / Remarks:

50% test material concentration

Results 3

Key result

Parameter:

SI

Value:

0.96

Test group / Remarks:

100% test material concentration

Any other information on results incl. tables

dose level	animal #	dpm	dpm minus background	average	std. dev.	SI
vehicle control	3601	915.72	841.11
	3602	1051.33	976.72
	3603	1408.1	1333.49
	3604	841.11	766.5
	3605	982.76	908.15	965.19	220.17
positive control (25% HCA in AOO)	3606	4533.36	4458.75
	3607	10722.62	10648.01
	3608	5412.3	5337.69
	3609	11221.56	11146.95
	3610	8755.23	8680.62	8054.4	3041.05	8.34
25% test substance in AOO	3611	1100.39	1025.78
	3612	1081.43	1006.82
	3613	1322.5	1247.89
	3614	1181.8	1107.19
	3615	1023.51	948.9	1067.32	115.76	1.11
50% test substance in AOO	3616	1448.66	1374.05
	3617	748.85	674.24
	3618	1581.27	1506.66
	3619	1342.65	1268.04
	3620	889.41	814.8	1127.56	363.16	1.17
100% test substance in AOO	3621	1234.49	1159.88
	3622	842.72	768.11
	3623	848.12	773.51
	3624	1084.95	1010.34
	3625	980.56	905.95	923.56	166.11	0.96

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Executive summary:

A dermal sensitization test was conducted with mice to determine the potential for n-butyl-3- hydroxybutyrate to produce sensitization after repeated topical applications. The test substance at 100% and two concentrations (50% and 25%) of the test substance in acetone/olive oil 4:1 v/v mixture (AOO) were topically applied to fifteen healthy test mice (5 mice/group) for three consecutive days. Three days after the last application, the mice were given a 20 J.LCi IV injection of 3H-methyl thymidine. Five hours later, all animals were euthanized via an overdose of inhaled Isoflurane and the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in disintegrations per minute per mouse (dpm/mouse). Each animal's ears were also evaluated for erythema and edema prior to each application and again on Day 6, prior to the IV injection. A vehicle control group (five animals) and a positive control group (five animals) were maintained under the same environmental conditions and treated in the same manner as the test animals. The vehicle control animals were treated with AOO and the positive control group animals were treated with a 25% w/w mixture of alpha~Hexylcinnamaldehyde (HCA) in AOO in the same manner as the test animals. These vehicle control and positive control group animals were shared with PSL Study Number 36785, which was conducted concurrently with this study. In accordance with Animal Welfare Practices. in an effort to reduce the total number of animals used, only one vehicle control group and one positive control group of animals was shared for the Sponsor's studies. The body weight data and dermal irritation scores from these shared vehicle control and positive control groups are included and presented in this report. However, the beta~ scintillation measured disintegrations ·per minute (DPM) values and Stimulation Index (SI) values were calculated independently for each individual study for these shared animal groups. A table summarizing the sensitization results noted is found below:

Mean DPM Stimulation Index (SI)

Group 1-Vehicle Control               965.19

Group 2 -Positive Control              8054.40                     8.34

Group 3 - 25% Test substance       1067.32                     1.11

Group 4- 50% Test substance        1127.56                     1.17

Group 5 - 100% Test substance       923.56                     0.96

Based on the results of this study, the test substance is not considered to be a contact dennal sensitizer. Proper conduct of the LLNA was confirmed via a positive response with 25% alpha~ Hexylcinnamaldehyde (HCA). a moderate contact sensitizer.