1-butylpyrrolidin-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 27 February 2015; Experimental Completion Date: 24 March 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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)) and in accordance with GLP standards (revised 1997, ENV/MC/CHEM(98)17) and (Directives 2004/9/EC and 2004/10/EC).

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-butylpyrrolidin-2-one

EC Number:

222-437-8

EC Name:

1-butylpyrrolidin-2-one

Cas Number:

3470-98-2

Molecular formula:

C8H15NO

IUPAC Name:

1-butylpyrrolidin-2-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): n-butyl pyrrolidone
- Substance type: organic
- Physical state: clear colorless liquid
- Purity test date: 2014-11-28
- Expiration date of the lot/batch: 01 March 2016
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.
- Storage condition of test material: room temperature, in the dark.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/Ca (CBA/Crl)
- Source: Charles River (UK), Kent, UK
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 15 to 23 g
- Housing: the animals were group housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) (ad libitum)
- Water (e.g. ad libitum): tap water (ad libitum)
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12.

IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

50%, 25% or 10% v/v

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using two mice, one mouse per test item concentration. The mice were treated by daily application of 25 цL of the test item at concentrations of 50% or 75% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) or two consecutive days (Days 1, 2) consecutively. The mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1 was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. The mouse treated at a concentration of 75% v/v in acetone/olive oil 4:1 was observed twice daily on Days 1 and 2 and once on Day 3. Local skin irritation was scored daily according to the scale of Draize. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1 was recorded on Day 1 (prior to dosing) and on Day 6. The body weight of the mouse treated at a concentration of 75% v/v in acetone/olive oil 4:1 was recorded on Days 1 and 3.
The thickness of each ear of the mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1 was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. The thickness of each ear of the mouse treated at a concentration of 75% v/v in acetone/olive oil 4:1 was recorded on Day 1.
Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
- Irritation: the test item would not produce systemic toxicity or excessive local irritation at a concentration of 50% v/v in acetone/olive oil 4:1.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µСі/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µСі to each mouse.
- Name of test method: Estimation of the Proliferative Response of Lymph Node Cells
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as "non-sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett's multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Results and discussion

Positive control results:

a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of 5.39 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

10% v/v in acetone/olive oil 4:1: 0.70 (negative).

Remarks on result:

other: see Remark

Remarks:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group

Results 2

Key result

Parameter:

SI

Value:

1.22

Test group / Remarks:

50% v/v in acetone/olive oil 4:1: 1.22 (negative)

Results 3

Key result

Parameter:

SI

Value:

0.91

Test group / Remarks:

25% v/v in acetone/olive oil 4:1: 0.91 (negative)

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Mean dpm/Animal (Standard Deviation) - Vehicle: Acetone/olive oil 4:1: 2814.18 (± 391.69); - Test Item 10% v/v in acetone/olive oil 4:1: 1957.70 (± 846.77); - Test Item 25% v/v in acetone/olive oil 4:1: 2556.20 (± 450.49); - Test Item 50% v/v in acetone/olive oil 4:1: 3441.98 (± 764.41); - Positive Control Item 25% v/v in acetone/olive oil 4:1: 15155.94** (± 2687.93). The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test item groups.

Any other information on results incl. tables

Preliminary Screening Test

Clinical observations, body weight and mortality data are given in Table 1 and local skin irritation is given in Table 2 (please see attached). The ear thickness measurements and mean ear thickness changes are given in Table 3 (please see attached).

The animal treated with the test item at a concentration of 75% v/v in acetone/olive oil 4:1 was found dead on Day 3. No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted in the animal treated at a concentration of 50% v/v in acetone/olive oil 4:1. Based on this information the dose levels selected for the main test were 50%, 25% and 10% v/v in acetone/olive oil 4:1.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per animal and the stimulation index are given in Table 4 (please see attached).

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment Group	Concentration	Stimulation Index	Result
Test Item	10% v/v in acetone/olive oil 4:1	0.70	Negative
	25% v/v in acetone/olive oil 4:1	0.91	Negative
	50% v/v in acetone/olive oil 4:1	1.22	Negative
Positive Control Item	25% v/v in acetone/olive oil 4:1	5.39	Positive

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5 (please see attached).

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body weight

Individual body weights and body weight change for test and control animals are given in Table 6 (please see atatched).

Body weight change of the test animals between Day 1 and Day 6 was comparable to those observed in the corresponding vehicle control group animals over the same period (please see tables attached).

Ear Thickness Measurements and Ear Thickness Changes, Local Skin Irritation

Ear Thickness Measurements and Mean Ear Thickness Changes are given in Table 7 and Local Skin Irritation in Table 8 (please see attached).

There was no increase in ear thickness (>25%) in any of the test or control animals on Days 3 and 6. No signs of irritation were seen in any of the test or vehicle control animals throughout the test (please see tables attached).

Ear Weight Measurements

Ear Weight Measurements are given in Table 9 (please see attached).

There was no increase in ear weight measurements (>25%) in any of the test or positive control animals on Day 6 (please see Tables 9 attached).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-sensitizer under the conditions of the test.
a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% v/v, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, a-Hexylcinnamaldehyde tech., 85%), at a concentration of 25% v/v in acetone/olive oil 4:1.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment Group	Concentration	Stimulation Index	Result
Test Item	10% v/v in acetone/olive oil 4:1	0.70	Negative
	25% v/v in acetone/olive oil 4:1	0.91	Negative
	50% v/v in acetone/olive oil 4:1	1.22	Negative
Positive Control Item	25% v/v in acetone/olive oil 4:1	5.39	Positive

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test. a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.