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Description of key information

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:
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
Qualifier:
equivalent or similar to
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
Qualifier:
equivalent or similar to
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)
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.
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.
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.
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.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable as the modified local lymph node assay was performed
Parameter:
SI
Remarks:
determined as cell count index
Value:
1.31
Test group / Remarks:
3 %
Parameter:
SI
Remarks:
determined as cell count index
Value:
1.89
Test group / Remarks:
10 %
Remarks on result:
other: exceeded the level of positivity
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.

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
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Caprolactam disulfide was tested in a GLP Guideline study (equivalent to OECD TG 429 and EU Method B. 46 ) 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.


Migrated from Short description of key information:
1) Vohr, 2011, NMRI mice, modified LLNA, sensitising

Justification for selection of skin sensitisation endpoint:
well documented GLP-guideline study equivalent to OECD TG 429 and EU Test Method B.46.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitization:

The test material does meet the criteria for classification and will require labelling for skin sensitisation in accordance with European regulation (EC) No 1272/2008 (Cat 1, H317).