1-butylpyrrolidin-2-one

Endpoint summary

Description of key information

- LLNA (OECD 429): Skin sensitisation_in vivo, LLNA, OECD Guideline 429, mice, result: non-sensitiser, 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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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:

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)

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.

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

Key result

Parameter:

SI

Value:

1.22

Test group / Remarks:

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

Key result

Parameter:

SI

Value:

0.91

Test group / Remarks:

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

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.

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).

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Key study:

Skin sensitisation potential of N-Butylpyrrolidone was assessed in a skin sensitisation study in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear (Harlan Laboratories Ltd, 2015). 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.

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

 

The test item was considered to be a non-sensitiser 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.

 

Disregarded study:

The skin sensitisation potential of N-Butylpyrrolidone was determined in the local lymph node assay in mice (LPT, 2013a; Report No. 29176). The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method. The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by a European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item.

However, this deviation of the procedure laid down in OECD 429 is quite substantial, as there it is specified, that each mouse should be carefully observed at least once daily for any clinical signs, either of local irritation at the application site or of systemic toxicity. Morever, the procedure described in test guideline OECD 429 does not allow a stop the test on day 4.

It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties. Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method (Ehling et al. 2005a and 2005b). Five concentrations of the test item (1 %, 5 %, 10 %, 25 % and 50 %, w/w) diluted with acetone/olive oil (3+1 v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. In addition, a positive control group (25 % solution v/v of α-hexyl cinnamic aldehyde in acetone/olive oil (3+1 v/v)) was employed.

In a pre-test 3 concentrations (10, 25 and 50 %) did not cause skin irriation.

In the main study treatment with the test item at concentrations of 1 % or 5 % did not reveal statistical significantly increased values for lymph node cell count and lymph node weight. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. Treatment with the test item at concentrations of 10 %, 25 % or 50 % (w/w) revealed statistical significantly increased values (p ≤ 0.01 or p ≤ 0.05) for lymph node cell count and lymph node weight. However, ear weights were also significantly increased at all 3 concentrations pointing to strongly irritating properties on the mouse ear. The stimulation indices of the lymph node cell count (sensitising properties) and ear weight (irritation properties) exceeded the threshold levels of 1.4 or 1.1. This is a clear contradiction to the results of the pre-test. The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In conclusion, under the present test conditions, test item at concentrations of 1 % or 5 % (w/w) in acetone/olive oil (3+1, v/v) did not reveal any sensitising properties in the local lymph node assay. Concentrations of 10 % or higher did not permit an evaluation due to the irritating properties of the test item in this test system as evident from the increased ear weight and an increase in ear thickness.

Taken together, the use of a custom method with no justification for guideline deviation, along with a blatant omission of important guideline requirements alone is enough to disqualify this study. When the profound lack of reproducibility between the range-finder and main study is also considered, the study can not be considered valid.

Justification for selection of skin sensitisation endpoint:
Reliability

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

N-Butylpyrrolidone is considered to be a non-sensitiser in LLNA and therefore does not need to be classified and labelled according to Regulation (EC) No 1272/2008 for this endpoint.