Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 30th, 2009 - December 3rd, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
A modification of the Local Lymph Node assay by measuring the cell counts instead of radioactive labeling was performed.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phthalimide
- Physical state: white solid
- Approval: until October 13, 2011
- Analytical purity: 99.8%
- Lot/batch No.: CHHPI301
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: Hsd Win:NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland, Horst, Netherland
- Age at study initiation: 8 weeks
- Weight at study initiation: 26 - 37 grams
- Housing: During the adaptation period up to 8 mice were housed together in conventional Makrolon type III cages. While during the study period the animals were single-housed in type II cages.

- Diet (e.g. ad libitum): The feed, PROVIMI KLIBA SA 3883 maintenance diet for rats and mice (from Provimi Kliba SA, CH-4303 Kaiseraugs, Germany)
- Water (e.g. ad libitum): 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

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.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0 (vehicle control), 2%, 10% and 50%.
No. of animals per dose:
6


Details on study design:
The test substance or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals.
This treatment was repeated on three consecutive days (d1, d2 and d3).

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated once at day 1 of the study.
The stability and the homogeneity of the test item in the vehicle was analytically verified for up to 4 days.

The test item or the vehicle were 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 25µL/ear.


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 heterogenous (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 toxico¬logical 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:
The last reliability test using Alpha Hexyl Cinnamic Aldehyde formulated in acetone/olive oil (4:1) at concentrations of 3%, 10% and 30% clearly showed the sensitizing potential of the test item.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Based on results obtained in validation studies and general experiences with this test system groups of mice were treated with vehicle, 2%, 10% or 50% Phthalimide in DMF. The NMRI mice did not show an increase in the stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item Phthalimide. The "positive level", which is 1.4 for the cell count index, was never reached or exceeded in any dose group.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable, as no radiolabel was used.

Any other information on results incl. tables

The "positive level" of ear swelling, which is 2x10-2 mm increase, i.e. about 10% of the control values, has not been reached or exceeded in any dose 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.

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

1. Direct LLNA (NMRI mice, female, 6 animals/group)

Dose (%) Weight index Cell count index

(index of mean +/- SD in %)

0* 1.00 +/- 19.79 1.00 +/- 26.55

2 1.04 +/- 8.99 0.97 +/- 14.76

10 1.03 +/- 25.13 0.90 +/- 34.13

50 1.08 +/- 21.21 1.07 +/- 23.29

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information There were no indications for a sensitizing potential of Phthalmide in a modified mouse LLNA.
Executive summary:

Lanxess (2009):

The modified Local Lymph Node Assay (IMDS) was performed in 24 female NMRI mice of the strain Hsd Win:NMRI (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item Phthalimide. The study was conducted according to OECD Guidelines No. 429 with the following test item concentrations: 0 (vehicle control), 2%, 10% and 50%. The test item was formulated in dimethylformamide (DMF) to yield a solution in the low and the mid concentration and a suspension in the high concentration. Compared to vehicle-treated animals, none of the parameters measured in the substance-treated groups, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay .These results show that there is no indication for a skin sensitizing effect after administration of a concentration of up to and including 50% Phthalimide in this test system. In conclusion, these results show that the test item Phthalimide has no sensitizing potential in mice after dermal application of up to and including a 50% concentration. In addition, no indication for a non-specific (irritant) activation was detected. Therefore, the concentration of 50% turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.