1,1'-dithiobis[hexahydro-2H-azepin-2-one]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-03-01 - 2011-03-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

measurement on d4 instead of d6, analysis of LLN weight & cell count, and ear weight & swelling

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

measurement on d4 instead of d6, analysis of LLN weight & cell count, and ear weight & swelling

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-dithiobis[hexahydro-2H-azepin-2-one]

EC Number:

245-910-0

EC Name:

1,1'-dithiobis[hexahydro-2H-azepin-2-one]

Cas Number:

23847-08-7

Molecular formula:

C12H20N2O2S2

IUPAC Name:

1-[(2-oxoazepan-1-yl)disulfanyl]azepan-2-one

Details on test material:

- Name of test material (as cited in study report): N,N' -Caprolactam-disulfid, (chemical name: (1,1' -Dithiobis {hexahydro-2H-azepin-2-one}))
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.32 % (Caprolactamdisulfid), (not used for calculation)
- Lot/batch No.: 19779/19777
- Expiration date of the lot/batch: December 14,2011
- Storage condition of test material: refrigerator

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: SPF-bred female NMRI mice of the strain Crl:NMRI BR
- Source: Charles River Germany Sulzfeld, Sandhofer Weg 7,97633 Sulzfeld, Germany
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 27 - 31 grams
- Housing: during the adaptation period up to 8 mice were housed together in conventional Makrolon type III cages, cages were changed at least twice a week. While during the study period the animals were single-housed in type II cages. Low-dust wood shavings named Lignocel BK 8-15 supplied by Rettenmaier & Sonne, GmbH & Co, 73494 Rosenberg, Germany were used as bedding. At the instigation of the Laboratory Animal Services (LAS) the wood granulate was analysed at random for contaminants. The relevant documents have been retained. The analytical results have not yielded evidence of any influence on the study objective.
- Diet (e.g. ad libitum): PROVIMIKLIBA SA 3883 maintenance diet for rats and mice (from Provimi Kliba SA, CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): tap water (drinking bottles) ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): about 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h, with artificial illumination

OTHER:
- the health status of the strains is checked regularly at random for the most important specific infection pathogens
- only healthy animals showing no signs of disease were used in the study.
- the animals were not vaccinated or treated with antiinfectives either before their arrival or during the adaptation or study period.
- the females were nulliparous and nonpregnant.

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

(A/OO), purity 85 %

Concentration:

Group 1: 0%(vehicle control),
Group 2: 3%,
Group 3: 10%,
Group 4: 30%
Group 5: positive control

No. of animals per dose:

6 animals/test item group and 6 control animals

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: modified LLNA
- Criteria used to consider a positive response: "positive level" = 1.4 for cell count indices, "positive level" of ear swelling = 2x10E2 mm increase, i.e. about 10 % of the control values

TREATMENT PREPARATION AND ADMINISTRATION:
- the test item was formulated, once before application in solvent.
- the formulations were visually described as solutions in the lowest concentration & as suspensions in the mid and in the highest concentration.
- during application the suspension was stirred on a magnet stirrer.
- stability and homogeneity of the test item in the vehicle was analytically verified for up to 4 days.
- 25 µL were applied to both ears of the mice

OTHER:
- the animals were identified by cage labels giving the test item, the animal number, dose, sex, and the study number and marking of the tail immediately before autopsy.
- the stability and the homogeneity of the test item in the vehicle was analytically verified for up to 4 days.

Positive control substance(s):

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

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control group(s; vehicle) by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogeneous (p<0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5 %. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99 % by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe's method, which according to Sachs can be used for both equal and unequal sample sizes. In this method of statistical processing of measurements a large number of comparisons is made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems the biological and toxicological relevance is also taken into consideration in the evaluation of statistical significance.
For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values were used in the evaluation of the biological relevance.

Results and discussion

Positive control results:

After treatment with Alpha Hexyl Cinnamic Aldehyde (group 5) the NMRI mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to control animals, which are of statistical significance. The "positive level", which is 1.4 for cell count indices has clearly been exceeded. The "positive level" of ear swelling, which is 2x10E2 mm increase, i.e. about 10 % of the control values, has been exceeded in the positive control group. Statistical significant increases of the ear weights and ear swelling compared to control animals were also detected for the positive control group. It has to be clarified that the "positive levels" mentioned above are exclusively defined for the NMRI outbreed mice used for this study, Such positive limits have to be calculated for each strain of mice individually.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The NMRI mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices of cell counts compared to control animals after application of the test item in the mid dose group, which are of statistical significance. The "positive level", which is 1.4 for cell count indices, has been exceeded in this group. The "positive level" of ear swelling which is 2x10E2 mm increase, i.e. about 10% of the control values, has been exceeded in the mid and the high dose groups. These changes are of statistical significance in these groups. A statistical significant increase in ear weights compared to control animals was detected for all dose groups. It has to be clarified that the "positive levels" mentioned above are exclusively defined for the NMRI outbreed mice used for this study, Such positive limits have to be calculated for each strain of mice individually.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: not applicable as the modified local lymph node assay was performed

Results 3

Parameter:

SI

Remarks:

determined as cell count index

Value:

1.31

Test group / Remarks:

3 %

Results 4

Parameter:

SI

Remarks:

determined as cell count index

Value:

1.89

Test group / Remarks:

10 %

Remarks on result:

other: exceeded the level of positivity

Results 5

Parameter:

SI

Remarks:

determined as cell count index

Value:

1.17

Test group / Remarks:

30 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : The NMRI mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices of cell counts compared to control animals after application of the test item in the mid dose group, which are of statistical significance. The level for positivity, which is 1.4 for cell count indices, has been exceeded in this group.

Any other information on results incl. tables

The body weights of the animals were not affected by any treatment.

Table 4: Results

	Tabular summary of the LLNA/IMDS results
1.	Direct LLNA (NMRI mice, female, 6 animals/group)
	Groups	Weight index (index of mean +/- SD in %)	Cell count index (index of mean +/- SD in %)
	Gr. 1	1.00 +/-19.28	1.00 +/- 10.99
	Gr.2	1.27 +/-18.13	1.31  +/- 28.60
	Gr.3	1.59↑+/-16.86	1.89↑+/-16.65
	Gr.4	1.20 +/-29.57	1.17 +/-35.03
	Gr.5	1.72↑+/- 8.75	1.98↑+/-16.63
2.	Ear swelling (NMRI mice, female, 6 animals/group, in 0.01 mm)
	Groups	day 1(mean +/- SD in %)	day 4 (mean +/- SD in %)	Index day 4
	Gr. 1	17.58  +/- 4.51	17.92  +/- 3.73	1.00
	Gr.2	17.67  +/- 4.41	19.50  +/- 6.38	1.09
	Gr.3	18.25  +/- 4.75	23.00↑+/- 16.27	1.28
	Gr.4	17.83  +/- 6.25	2S.17↑+/-13.00	1.40
	Gr.5	18.17  +/- 3.95	20.92↑+/- 6.90	1.17
	↑= statistically significant increase (p<_0.05)
3.	Ear weight (NMRI mice, female, 6 animals/group,in mg per 8 mm diameter punch)
	Groups		day 4 (mean +/- SD in %)	Index day 4
	Gr. 1		12.67 +/- 5.81	1.00
	Gr.2		15.23↑ +/- 12.29	1.20
	Gr.3		19.58↑ +/- 20.91	1.55
	Gr.4		18.53↑ +/- 1.35	1.46
	Gr.5		15.66↑ +/- 7.55	1.24
	↑= statistically significant increase (p<_0.05)

Table 5: Individual cell counts

Individual cell counts
Cell counts [Thousand cells / mL cell suspension] in auricular lymph nodes* on study day 4 after daily epicutaneous application of a 0,3, 10 or 30 % solution or suspension of test item or Alpha Hexyl Cinnamic Aldehyde in acetone/olive oil for 3 days onto the ears of female NMRI mice (6 / treatment group) in a Local Lymph Node Assay
Treatment group	Animal no.	Cell count [Thousand cells/mL lymph node suspension					Cell count index**
		Repeat determination	Arithmetic mean	Group mean	SD	SD [%]
Vehicle control (Group 1)	1	10870 / 10931	10900.5	11433.33	1256.10	10.99	1.00
	2	9998 / 10129	10063.5
	3	12283 / 12201	12242
	4	10168 / 9937	10052.5
	5	13105 / 12696	12900 5
	6	12314 / 12568	12441
Group 2	7	19753 / 19722	19737.5	14971.17	4281.41	28.60	1.31
	8	9460 / 9337	9398.5
	9	12669 / 12469	12569
	10	15133 / 14660	14896.5
	11	20070 / 20465	20267.5
	12	12984 / 12932	12958
Group 3	13	23917 / 24068	23992.5	21686.00	3607.47	16.65	1.89
	14	23344 / 23683	23513.5
	15	26143 / 26513	26328
	16	16373 / 16326	16349.5
	17	20064 / 20066	20065
	18	19703 / 19792	19747.5
Group 4	19	14547 / 14366	14456.5	13322.76	4667.18	35.03	1.17
	20	7569 / 7253	7411
	21	20454 / 21052	20753
	22	10373 / 10218	10295.5
	23	11400 / 11562	11481
	24	15538 / 15541	15539.5
Group 5	25	20071 / 19856	19963.5	22912.76	3761.35	16.63	1.98
	26	19868 / 19543	19705.5
	27	18854 / 18775	18814.5
	28	23242 / 22955	23098.5
	29	27918 / 27774	27846
	30	26352 / 26145	26248.5
* : The right and left auricular lymph node of each animal was obtained, crushed trough a sieve and dispersed in 2 mL phosphate buffered saline. An aliquot of this sample was taken for a repeated determination of the cell count per mL of this cell suspension. The cell count of every animal is the calculated mean of these two measurements.
**: Cell count Index: mean cell count of the animals of a treatment group divided by the mean cell count of the vehicle control group. The Cell count index for the vehicle control group is 1.

Table 6: Body weights

Body weights
Animal-No.	Body weight in g
	Day l	Day4
group1
1	28	27
2	30	32
3	31	31
4	30	30
5	28	29
6	30	31
Mean	29.5	30.0
group2
7	31	33
8	29	30
9	29	31
10	28	28
11	29	29
12	29	29
Mean	29.2	30.0
group3
13	30	31
14	31	32
15	28	30
16	28	29
17	29	32
18	27	30
Mean	28.8	30.7
group4
19	30	31
20	28	29
21	29	31
22	30	32
23	29	29
24	28	29
Mean	29.0	30.2
group5
25	28	28
26	29	29
27	30	30
28	29	30
29	28	30
30	29	29
Mean	28.8	29.3

Applicant's summary and conclusion

Interpretation of results:

other: moderate skin sensitizer

Remarks:

Criteria used for interpretation of results: other: EU-GHS

Conclusions:

The GLP-study was performed equivalent to the OECD Guideline 429, EU Method B.42, modified local lymph node assay in mice and is considered to be reliable without restrictions (reliability Klimisch 1). The test material did induce a response and the results show that the test item may have a moderate sensitising potential in mice after dermal application of a 10 % concentration with an EC value of 4.09 %. The test material was considered to be sensitising under the conditions of the test. Additionally the concentration of 3 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitisation.

Executive summary:

The test item was tested in a GLP Guideline study (equivalent to OECD TG 429, EU method B. 42) for its sensitising potential in a modified local lymph node assay in female NMRI mice (Vohr, 2011). The aim of these investigations was to establish whether there is any specific (sensitising) or non-specific (irritant) stimulating potential of the test item. The test item was applied epicutaneously for 3 consecutive days onto both ears of the animals in concentrations of 0 %, 3 %, 10 % and 30 % (solved in acetone / olive oil 4:1 w/v). The results of the positive control substance Alpha Hexyl Cinnamic Aldehyde verified the validity of the test. After treatment there was a clear increase compared to control animals regarding the weights of the draining lymph nodes, which is of statistical significance in the mid dose group. A sensitising potential can be assumed from the increases in cell proliferation in the draining lymph nodes. On the basis of our experiences using this method the "positive level" had been set to an increase in cell count index by 0.4 (i.e. index \ 1.4), which has been exceeded in the mid dose group. Differentiation indices (DI) calculated which is the quotient of the relative lymph node reaction divided by the relative acute skin reaction was < 1 for the mid concentration tested, i. e. 0.79. This DI value does point to an irritating potential of the test item. The "positive level", which is 1.4 for cell counts has been exceeded in the mid dose group. The "positive level" of ear swelling which is 2x10E2 mm increase, i.e. about 10% of the control values, has been exceeded in the mid and the high dose group. These changes are of statistical significance in the mid and the high dose groups. A significant increase compared to vehicle treated animals regarding ear weights was detected in all dose groups. An increase in this parameter would point to an acute irritating (inflammatory) response. Due to the strong irritant property of the test item at this concentration it is not quite clear if the cell proliferation is exclusively induced by this non-specific (inflammatory) reaction. Clarification can only be reached by a secondary response assay, and a sensitising potential cannot fully be excluded by the data available so far. So this study does point to a non-specific (irritant) and to a specific immunostimulating (sensitising) potential of the test item. In conclusion, these results show that the test item may have a moderate sensitising potential in mice after dermal application of a 10 % concentration with an EC value of 4.09% ( in accordance with the classification proposed in the Technical Report No. 78 of the ECETOC). Therefore, the concentration of 3 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitisation.