Registration Dossier

Administrative data

Description of key information

The test item was assessed for both its skin sensitizing and irritant potential in a modified Local Lymph Node Assay with the following concentrations: 0 % (vehicle control), 2 %, 20 % and 100 %.

The test item was formulated in acetone / olive oil 4:1 to yield a solution. Compared to vehicle (A/OO) treated animals there were increases regarding the cell counts and the weights of the draining lymph nodes in all dose groups, which are of statistical significance. In addition, the "positive level" of the stimulation index for the cell counts, which is 1.4, was exceeded in all dose groups. The “positive level”, indicating irritating potential of the test item, which is 1.10 for ear swelling index, was statistically significantly exceeded in the high dose group. Therefore, an indication for a specific skin sensitizing effect after administration of a concentration >= 2 % test item is revealed in this test system, and also an indication for an unspecific irritant potential of the undiluted test item. Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 1%. In accordance with the classification proposed in the Technical Report No. 87 of the ECETOC this value corresponds to a strong skin sensitizer. The validity of the assay was demonstrated by the positive results of the positive control group (40 % Alpha Hexyl Cinnamic Aldehyde).

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:
Nov./Dec. 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Although the experiment was conducted until the beginning of december the study was initiated and assigned before the revision of the stand information requirements (ANNEX VII) of REACH regulation comes into force. There are sound scientific and practical reasons why valeric acid anhydride was investigated in the LLNA. Valeric anhydride is suspected to be a skin sensitizer (see ANNEX IIII Inventory). Recently the methylester of valeric acid (= pentanoic acid) and the anhydrides of trimethyl acetic acid and heptanoic acid showed a skin sensitizing potential in the LLNA. In addition several publications questioned if anhydride could be predicted correctly by in vitro assays. On that background and having in mind that no subcategorisation (= conclusion on its potency) would be possible if the outcome of in vitro tests points towards that valeric anhydride is a skin sensitizer, the LLNA was the only way to come to a correct assessment of the skin sensitising potency of valeric anhydride. A more important aspect is that the collection of comparable studies for a category (aliphatic acid anhydride) is enlarged which facilites the assessment of category members with data lacking.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- modified LLNA (IMDS): Measurement of cell counts instead of radioactive labeling. In addition, measurements of ear swelling and ear weights were done to discriminate the irritating potential from the sensitizing potential of the test substance.
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). By comparing the specific immune reaction induced by the test item in the draining lymph nodes (cell counts / lymph node weights) with the immediate nonspecific acute skin reaction (ear swelling / ear weight), it is possible to differentiate the irritant potential from the sensitizing potential of the compound tested .Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Detailed Information on validation of IMDS, scientific justification, method and assessment of results are given in:

- Homey, B., von Schilling, C., Blümel, J., Schuppe, H.-C., Ruzicka, T., Ahr, H.-J., Lehmann, P. and Vohr, H.-W.: An integrated Model for the Differentiation of Chemical-Induced Allergic and Irritant Skin Reactions (IMDS). Toxicol. and Appl. Pharmacol. 153, 83-94 (1998).
- Vohr, H.-W., Blümel, J., Blotz, A., Homey, B. and Ahr, H.J. An intra-laboratory validation of IMDS: Discrimination between (Photo) Allergic and (Photo) Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509 (2000).
- ECETOC Technical Report No. 87, Brussels (2003)
- Ehling G., Hecht M., Heusener A., Huesler J., Gamer A.O., v. Loveren H., Maurer Th., Riecke K., Ullmann L., Ulrich P., Vandebriel R., Vohr H.-W. An European Inter-Laboratory Validation of Alternative Endpoints of the Murine Local Lymph Node Assay. 2nd ROUND.Toxicology 212 (2005), 69-79
- Gamer A.O., Nies E., Vohr H.-W.: Local Lymph Node Assay (LLNA): Comparison of different protocols by testing skin-sensitizing epoxy resin system components. Reg. Tox. Pharmacol. 52 (2008), 290-298.


GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Stability under test conditions: The stability of the test item in the vehicle was analytically verified for up to 4 days. The test item formulations in the vehicle were visually described as solutions.
Species:
mouse
Strain:
other: Crl: NMRI BR
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl: NMRI BR
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 28-33 g
- Housing: During the study period the animals were single-housed in Makrolon type II cages.
- Diet and water: ad libitum
- Acclimation period: at least 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 -70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 (vehicle control), 2%, 20% and 100%
No. of animals per dose:
six
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated once before application in A/OO. The formulation was applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance. For negative and positive control a dose group treated only with the vehicle A/OO and one treated with hexyl cinnamic aldehyde in A/OO, respectively, in the above described manner was used.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling
- ear weight
- body weights
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The individual values from actively treated groups were compared with those from the control group. A pre-testing was carried out by a Cochran test. Furthermore, depending on the statistical result, a Dunnett test significance test was conducted (significance levels of 5 %; two-tailed).
In this method of statistical processing of measurements a large number of comparisons are 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 for the final evaluation of the biological relevance
All individual data were recorded and archived also in the cases where individual data were not reported, e.g. ear swelling and ear weight.
Positive control results:
Positive control - sensitizing potential: After treatment with Alpha Hexyl Cinnamic Aldehyde the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals, which are of statistical significance. The "positive level", which is 1.4 for cell count indices, clearly was exceeded (value: 1.64).
Positive control - irritating potential: The "positive level" of ear swelling, which is 2x10-2 mm increase, i.e. about 10 % of the control values, was exceeded in the positive control group. This increase was of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was also detected.
Parameter:
other: EC1.4
Value:
> 0 - < 1
Test group / Remarks:
Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 1%.
Remarks on result:
other: The concentration (%) inducing SI of 1.4 = cut-off value for the cell count index for NMRI outbred mice.
Parameter:
SI
Value:
1
Remarks on result:
other: cell cout indices of vehicle control
Parameter:
SI
Value:
2
Remarks on result:
other: cell count indices of 2% test item treated group: The positive level, which is 1.4 for cell count indices, was exceeded in this group.
Parameter:
SI
Value:
3.83
Remarks on result:
other: cell count indices of 20% test item treated group: The positive level, which is 1.4 for cell count indices, was exceeded in this group.
Parameter:
SI
Value:
4.02
Remarks on result:
other: cell count indices of 100% test item treated group:The positive level, which is 1.4 for cell count indices, was exceeded in this group.

Table 1: Summary of the LLNA/IMDS results (means of 6 animals per group)

Parameter investigated

Vehicle

control

Positive control

Dose 2 %

 Dose 20 %

Dose 100 %

Stimulation index:

weight of draining lymph nodes

1.00

 1.86*

1.54*

3.15*

3.93*

Stimulation index:

cell count in draining lymph nodes

1.00

 2.23*

2.00*

3.83*

4.02*

Ear swelling in 0.01 mm on day 4 (index)

18.17

(1.00)

 20.08*

(1.11)

19.08*

(1.05)

19.33*

(1.06)

23.08*

(1.27)

Ear weight in mg / 8 mm diameter punch on day 4 (index)

12.87

(1.00)

 15.29*

(1.19)

13.91*

(1.08)

15.93*

(1.24)

18.40*

(1.43)

* statistically significant increase (p <= 0.05)

Test item - sensitizing potential: The NMRI mice did show an increase in stimulation indices for cell counts, which is of statistical significance (see Table1), and for weights of the draining lymph nodes after application of the test item at 2 %, 20 % and 100 %. In addition, the "positive level", which is 1.4 for cell count indices, was exceeded in these groups ( (value group 2%: 2.00; value group 20%: 3.83; value group 100%: 4.02).

Test item - irritating potential: The “positive level”, indicating irritating potential of the test item, which is 1.10 for ear swelling index was statistically significantly exceeded in the high dose group (value: 1.27). In the low and mid dose group the ear swelling are statistical significant increased. A significant increase compared to vehicle treated animals regarding ear weights was detected in the mid and high dose group.

Positive control - sensitizing potential: After treatment with Alpha Hexyl Cinnamic Aldehyde (Positive Control) the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts (see Table 1) compared to vehicle control animals. The "positive level", which is 1.4 for cell count indices, clearly was exceeded (value: 2.23).

Positive control - irritating potential: The "positive level" of ear swelling, which is 1.10 for ear swelling index, was exceeded in the positive control group. This increase was of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was also detected. The body weights of the animals were not affected by any of the treatment.

Interpretation of results:
other: sensitising
Executive summary:

The test item was assessed for both its skin sensitizing and irritant potential in a modified Local Lymph Node Assay with the following concentrations: 0 % (vehicle control), 2 %, 20 % and 100 %.

The test item was formulated in acetone / olive oil 4:1 to yield a solution. Compared to vehicle (A/OO) treated animals there were increases regarding the cell counts and the weights of the draining lymph nodes in all dose groups, which are of statistical significance. In addition, the "positive level" of the stimulation index for the cell counts, which is 1.4, was exceeded in all dose groups. The “positive level”, indicating irritating potential of the test item, which is 1.10 for ear swelling index, was statistically significantly exceeded in the high dose group. Therefore, an indication for a specific skin sensitizing effect after administration of a concentration >= 2 % test item is revealed in this test system, and also an indication for an unspecific irritant potential of the undiluted test item. Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 1%. In accordance with the classification proposed in the Technical Report No. 87 of the ECETOC this value corresponds to a strong skin sensitizer. The validity of the assay was demonstrated by the positive results of the positive control group (40 % Alpha Hexyl Cinnamic Aldehyde).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I the test item shall be classified as skin sensitiser (Category 1A)