4,4'-biphthalic dianhydride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-17 to 2016-04-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 22, 2010

Deviations:

yes

Remarks:

Measurement of the cell proliferation by cell counting 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.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

amended in Commision Regulation (EU) No. 640/2012, published in the Official Journal of the European Union L193, dated July 20, 2012.

Principles of method if other than guideline:

Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and the Note of
Guidance SWP/ 2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Ehling G et al., Toxicology 212, 60-68
and 69-79 (2005).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-biphthalic dianhydride

EC Number:

219-342-9

EC Name:

4,4'-biphthalic dianhydride

Cas Number:

2420-87-3

Molecular formula:

C16H6O6

IUPAC Name:

5-(1,3-dioxo-1,3-dihydro-2-benzofuran-5-yl)-1,3-dihydro-2-benzofuran-1,3-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

4,4'-biphthalic dianhydride supplied by Evonik Fibres GmbH (Lenzing, Austria), batch 20130801 (2013), purity 99.7 % w/w

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: 
- Species: Mice (non-pregnant, nulliparous, healthy)
- Starin: NMRI / Crl:NMRI
- Source: Charles River Laboratories Germany GmbH, 97633 Sulzfeld, Germany
- Sex: female
- Age: 63 days
- Weight at study initiation: 25 - 34 g
- Housing: single
- Diet: ad libitum, ssniff R/M-H V 1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimatisation period: at least 5 days
- Controls: 6 female mice, treated with vehicle
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3 °C
- Humidity (%): 55 % +/- 15 %
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Three concentrations of test item: 10%, 25% and 50%, suspended in acetone/olive oil (4:1, v/v) (w/w)
Vehicle control group (acetone/olive oil (4:1, v/v))
Positive control group (20% solution (v/v) of a-hexyl cinnamic aldehyde in acetone/olive oil (4:1, v/v))

No. of animals per dose:

6 female mice

Details on study design:

PREPARATION OF TEST ITEM
- The test item was suspended in acetone /olive oil (4:1, v/v).

PRE-SCREEN TESTS:
- 3 animals/test item to examine irritating potential and handling/application to select the appropriate concentrations. Three concentrations of 10%, 25% and 50% suspended in acetone /olive oil (4:1, v/v) were examined. Doses were selected according to OECD guideline from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc.
the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc
- Test item was a yellow/brown powder. Hence, a 50% suspension was the highest feasible concentration in acetone / olive oil (4:1, v/v).

TREATMENT PREPARATION AND ADMINISTRATION:
- Three concentrations of 10, 25 and 50% of test item in acetone /olive oil (4:1, v/v) were examined..
- Vehicle test item: acetone /olive oil (4:1, v/v)
- Vehicle positive control: acetone /olive oil (4:1, v/v)
- Positive control: 25 µL of a 20% solution of a-hexyl cinnamic aldehyde
- Weight of each animal, clinical observations and ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
- Open application of the test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear:
vehicle alone
test item in 3 concentrations
positive control
- This treatment was repeated on three consecutive days (d1, d2 and d3).
- 24 h after last application (day 4), ear swelling measurements are carried out at the helical edge of both ears using an Oditest micrometer
- Immediatly after measurement animals were sacrificed under ether anaesthesia.
The appropriate organs were then removed.
Lymphatic organs (the auricular lymph nodes) were then stored on ice in PBS /0.5% BSA.
Investigations:
- ear swelling
- ear weight (Punch biopsies of 8 mm of the apical area)
- 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.
Animals were observed once daily for clinical signs of local systemic irritation at the application site or of systemic toxity. Body weight were recorded on day 1 and day 4.

Positive control substance(s):

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

Statistics:

The so-called stimulation (or LLN-) indices to determine the sensitising potential were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. U-test was performed for cell count, too. Outliers were determined according to the Nalimov test.
In addition, the acute inflammatory skin reaction (irritating potential) was 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 employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.
The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for the ear weight stimulation index was set at 1.1.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.051

Test group / Remarks:

Group 2, 10%; Lymph node cell

Remarks on result:

other: detailed results see below

Results 2

Key result

Parameter:

SI

Value:

1.036

Test group / Remarks:

Group 3, 25%; Lymph node cell

Remarks on result:

other: detailed results see below

Results 3

Key result

Parameter:

SI

Value:

1.077

Test group / Remarks:

Group 4, 50%; Lymph node cell

Remarks on result:

other: detailed results see below

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
The animals were euthanized by carbon dioxide (CO2) inhalation and laparotomised.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS /0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

DETAILS ON STIMULATION INDEX CALCULATION
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 interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

EC3 CALCULATION
The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation.

CLINICAL OBSERVATIONS:
Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal.

BODY WEIGHTS
The weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

Any other information on results incl. tables

Main study results: Stimulation indices (SI):

Parameter	Group 1, negative control	Group 2, 10%	Group 3, 25%	Group 4, 50%	Group 5, positive control
Lymph node cell count	1.000	1.051	1.036	1.077	1.671
Lymph node weight	1.000	0.978	1.087	1.130	1.935
Ear weight	1.000	0.923	0.950	0.961	1.116
Ear thickness, TD4	1.000	1.004	1.047	1.043	1.226

____ significantly different from control at p ≤ 0.01

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions, test item at concentrations of 10%, 25% or 50% (w/w) in acetone/olive oil (4:1, v/v) did not reveal any sensitising properties in the local lymph node assay.

Executive summary:

The purpose of this study was to determine the sensitising potential of test item in the modified local lymph node assay in mice.

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 nodecell count to assess proliferation has been established by an 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. 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 nodecell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Three concentrations of test item (10%, 25% and 50%), suspended inacetone/olive oil (4:1, v/v)(w/w), were tested in six female NMRI mice per group and compared to a vehicle control group (acetone/olive oil (4:1, v/v)).

Acetone/olive oil (4:1, v/v) was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear at such high concentrations.

A 50% suspension was the highest feasible concentration of test item in acetone/olive oil (4:1, v/v).

In addition, a positive control group (20% solution (v/v) of a-hexyl cinnamic aldehyde inacetone/olive oil (4:1, v/v)) was employed.

Stimulation indices (SI):

Parameter	Group 1, negative control	Group 2, 10%	Group 3, 25%	Group 4, 50%	Group 5, positive control
Lymph node cell count	1.000	1.051	1.036	1.077	1.671
Lymph node weight	1.000	0.978	1.087	1.130	1.935
Ear weight	1.000	0.923	0.950	0.961	1.116
Ear thickness, TD4	1.000	1.004	1.047	1.043	1.226

____ significantly increased compared to control at p ≤ 0.01

In the main study treatment with test item at concentrations of 10%, 25% or 50% in acetone/olive oil (4:1, v/v)(w/w) did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation indices calculated for the lymph node cell count did not exceed the threshold level of 1.4. In addition, no increase in the lymph node weight was observed. Hence, the test item is classified as not sensitising.

The threshold level of 1.1 for the ear weight stimulation index was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted.

The positive control group caused the expected increases in lymph node cell countand lymph node weight (statistically significant atp ≤ 0.01). Therefore, the study can be regarded as valid.

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 10%, 25% or 50% (w/w) in acetone/olive oil (4:1, v/v) did not reveal any sensitising properties in the local lymph node assay.