2-butoxyethyl benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 429, EPA OPPTS 870.2600, EU Method B.42 and in accordance with the Principles of Good Laboratory Practices (GLP)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-butoxyethyl benzoate

EC Number:

226-685-8

EC Name:

2-butoxyethyl benzoate

Cas Number:

5451-76-3

Molecular formula:

C13H18O3

IUPAC Name:

2-butoxyethyl benzoate

Test material form:

other: variable colured liquid

Details on test material:

- Name of test material (as cited in study report): Butyl Cellosolve™ Benzoate
- Physical state: variable coloured liquid
- Analytical purity: 100% 2-butoxyethyl benzoate (per MSDS)
- Lot/batch No.: 02112012-JLT
- Expiration date of the lot/batch: 11 February 2013
- Storage condition of test material: Ambient (+18 to +36 ºC)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratory, Bar Harbor, ME
- Age at study initiation: young adults - Preliminary Animals - 10-11 weeks; Test and Control Animals - 9-10 weeks
- Weight at study initiation: Preliminary Animals - 18.4-23.4 grams; Test and Control Animals - 14.2-21.3 grams at experimental start.
- Housing: individually housed
- Diet (ad libitum): Harlan Teklad Certified Global 16% Protein Rodent Diet® #2016C
- Water (ad libitum): Filtered tap water was supplied ad libitum
- Acclimation period: 15-21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23 °C
- Humidity (%): 46-52%
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

The sample was soluble in and dosed as a w/w mixture in methyl ethyl ketone (Fisher Scientific, Lot #121215, Exp. Date: 3/31/2017) and tested at concentrations of 25%, 50% and undiluted (100%) - preliminary study and test substance concentrations of 100% (undiluted), and 5% and 25% w/w mixtures in methyl ethyl ketone were selected for testing in the LLNA main study

No. of animals per dose:

Number of Animals: 33
Number of Groups: 9
Number of Animals per Group:
Preliminary Irritation (4 groups): 2 per group
Test (3 groups): 5 per group
Vehicle control and positive control animals:
The number of animals in these groups was as follows: Vehicle (Negative) Control Group (1 group): 5 per group
Positive Control Group (1 group): 5 per group
Groups unable to complete testing due to Hurricane Sandy
Test (3 groups): 5 per group
Vehicle (Negative) Control Group (1 group): 5 per group
Positive Control Group (1 group): 5 per group
Sex: Female, nulliparous and non-pregnant.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test substance was soluble in and dosed as a w/w mixture in methyl ethyl ketone (Fisher Scientific, Lot #121215, Exp. Date: 3/31/2017).
- Three test substance concentrations and the vehicle control were used for preliminary toxicity testing. The undiluted test substance (100%), and concentrations of 50% and 25% of the test substance in methyl ethyl ketone were tested to determine the highest concentration to avoid overt systemic toxicity and excessive local irritation. Each group consisted of two mice. The ears of each mouse were scored for erythema and edema prior to dosing on Days 1, 2, 3, and prior to termination on Day 6 according to the scoring system.
Twenty-five microliters (25 µl) of the appropriate dilution of the test substance concentration or the vehicle alone was applied to the dorsum of both ears of each mouse (50 µl total) for three consecutive days (Days 1, 2 and 3). Application was done using an appropriate size micropipette to accurately deliver 25 µl. 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 Days 1, 2, 3 and 6, each site was evaluated for local irritation (erythema & edema).
In addition, duplicate measurements of the thickness of each animal’s ears were made using a micrometer (Digital Mitutoyo Micrometer, SN: 220468). The measurements were made at the apex of the pinna. Measurements were taken on the preliminary screen animals on Day 1 (predose), Day 3 and Day 6. Increases in ear swelling on Days 3 and 6 were calculated for each animal relative to the thickness measurement taken on Day 1.
Animals were observed daily for signs of toxicity and body weights were recorded. Ear swelling and erythema were used to identify concentrations of the test material that produce irritation to the ears of mice.
Based on the results of the preliminary screen, test substance concentrations of 100% (undiluted), and 5% and 25% w/w mixtures in methyl ethyl ketone were selected for testing in the LLNA main study

MAIN STUDY
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) - Group 6 - Vehicle control; Group 7 - Positive control substance (25% HCA); Group 8 - Test substance - 5%; Group 9 - Test substance 25% and Group 10 - 100%. Dilutions of the test substance (5% and 25%) were prepared as w/w mixtures in methyl ethyl ketone. A single concentration of a 25% w/w mixture of HCA in methyl ethyl ketone was prepared as a positive control. All dosage preparations were freshly prepared on the day of administration. Beginning on Day 1, a volume of 25 µl of the vehicle, the appropriate test substance concentration, or the positive control substance was applied to the dorsum of both ears of each mouse (50 µl total) once per day for three consecutive days (Days 1, 2, and 3) using a micropipette. During application, the material was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette. On Day 6 of the study (three days after the final topical application) 250 µl of sterile phosphate buffered saline (PBS, Sigma-Aldrich, Batch #: SLBC5574, Exp. Date: 8/20/13) containing 20 µCi of 3H-methyl thymidine (Lot #201210) was injected intravenously via the tail vein of each mouse. Approximately five hours after the injection, all animals were euthanized and the draining auricular lymph nodes were excised. The lymph nodes were evaluated for each individual mouse. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently massaging the lymph nodes between the frosted ends of two microscope slides over a collection vessel. The slides were then rinsed briefly with PBS into the vessel. The contents of the vessel were transferred to a centrifuge tube and washed with an excess of PBS and centrifuged for approximately 10 minutes at 1800 rpm, with a RCF8 of 489G. This process was carried out twice. In both cases, the supernatant was decanted and discarded following each centrifugation. After the second wash, 5 mL of 5% trichloroacetic acid in distilled water (TCA, Fisher Scientific, Lot #: 114546, Exp. Date: 9/2016) was then added to the sediment and the tube was vortexed briefly. The DNA was then precipitated in the 5% TCA at approximately 3.5-3.9 C overnight (approximately 18 hours). Following the overnight precipitation of the DNA, the tubes were centrifuged again for approximately 10 minutes and the supernatant was discarded. The resulting precipitate was resuspended using 1 mL of the 5% TCA and transferred to 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine was measured by beta-scintillation counting and expressed as disintegrations per minute, minus background dpm.

Positive control substance(s):

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

Statistics:

Statistical analysis was performed on the body weight, body weight gain and dpm values. Significance was judged at p <0.05. The treated group and vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to the vehicle control group by Dunnett’s t-test for multiple comparisons. Where variances were 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:

Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer, which elicited a proliferative response with a SI value of 3.07 relative to the vehicle control.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Treatment of mice with 5%, 25% and 100% of 2-butoxyethyl benzoate resulted in stimulation index values of 1.04, 0.99 and 0.97, respectively, relative to vehicle control mice.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Following were the mean dpm values noted - Group 6 - Vehicle control - 1011.11; Group 7 - Positive control - 3104.07; Group 8 - 5% - 1054.0; Group 9 - 25% - 1002.92; Group 10 - 981.75

Any other information on results incl. tables

Preliminary study -

A preliminary screening study was conducted with the undiluted test substance (100%), and concentrations of 25% and 50% w/w mixtures in methyl ethyl ketone and the vehicle alone. On Days 1, 2, 3 and 6, each dose site was evaluated for local irritation. All mice appeared active and healthy and there were no consistent treatment-related effects on body weight. There was no dermal irritation observed in the vehicle control group. Very slight erythema (score = 1) was seen at the 100%, 50% and 25% concentrations on Day 3. Desquamation was present at the dose sites of both mice dosed with 100% on Day 6. Excessive local skin irritation is indicated by an

erythema score ≥3 on any day of measurement and/or an increase in ear thickness of ≥25% on Day 3 or 6 relative to the measurement on Day 1. None of the concentrations tested produced an increase in ear thickness greater than 17%. Therefore, the 100% concentration was chosen as the top dose for the main study, along with 25% and 5% to characterize the dose response.

Main study -

All mice appeared active and healthy, and there were no consistent treatment-related effects on body weight over the course of the study. There was no dermal irritation (based on erythema observations) noted for the treatment group at the 5% concentration. Very slight erythema (score = 1) was observed at one site on Day 3 for the 25% concentration.For the 100% treatment group, very slight erythema (score = 1) was evident for some mice

between Days 2 and 6, with desquamation present at all sites on Day 6.

No dermal irritation was observed at any site in the vehicle control group (Group 1). Very slight erythema (score = 1) and desquamation were noted for all sites and slight edema (score = 1) was evident at three sites in the positive control group (Group 2).

Test groups	Mean DPM	Stimulation Index
Group 6 - Vehicle control	1011.11	-
Group 7 - Positive control	3104.07	3.07
Group 8 - 5% test substance	1054.10	1.04
Group 9 - 25% test substance	1002.92	0.99
Group 10 - 100% test substance	981.75	0.97

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the study, as a stimulation index (SI) of less than 3.0 was observed in all treatment groups, 2-butoxyethyl benzoate was considered negative for dermal sensitization potential in the LLNA. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.

Executive summary:

A dermal sensitization test was conducted with female mice to determine the potential for 2-butoxyethyl benzoate to produce dermal sensitization after repeated topical applications.

Three concentrations (5% and 25% in methyl ethyl ketone and 100% (undiluted, as received)) of the test substance were topically applied to fifteen healthy female test mice (5 mice/group) for three consecutive days. Three days after the last application, the mice were given a 20 μCi IV injection of 3H-methyl thymidine. Five hours later, 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 female mice) and a positive control group (five female mice) were maintained under the same environmental conditions and treated in the same manner as the test animals. The vehicle control animals were treated with methyl ethyl ketone and the positive control animals were treated with a 25% w/w mixture of alpha-Hexylcinnamaldehyde Technical (HCA) in methyl ethyl ketone in the same manner as the test animals.

Test groups	Mean DPM	Stimulation Index
Group 6 - Vehicle control	1011.11	-
Group 7 - Positive control	3104.07	3.07
Group 8 - 5% test substance	1054.10	1.04
Group 9 - 25% test substance	1002.92	0.99
Group 10 - 100% test substance	981.75	0.97

Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer. As a stimulation index (SI) of less than 3.0 was observed in all treatment groups, 2-butoxyethyl benzoate was considered negative for dermal sensitization potential in the LLNA according to the Guidance to Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.