Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane- l,2-diol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

5-Isobenzofurancarboxylic acid, 1,3-dihydro-1,3-dioxo-, reaction products with 1,2-propanediol

IUPAC Name:

5-Isobenzofurancarboxylic acid, 1,3-dihydro-1,3-dioxo-, reaction products with 1,2-propanediol

Constituent 2

Reference substance name:

5-Isobenzofurancarboxylic acid, 1,3-dihydro-1,3-dioxo-, polymer with 1,2-propanediol

EC Number:

613-407-9

Cas Number:

63948-88-9

IUPAC Name:

5-Isobenzofurancarboxylic acid, 1,3-dihydro-1,3-dioxo-, polymer with 1,2-propanediol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

ZAN 573. Lot 130072591. yellow to brown course powder. molecular weight: 424.314 g/m. 100% purity. Expiry date: 24 January 2016.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/J Rj mice, ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 9 weeks
- Weight at study initiation: 19.8-21.9 g
- Housing: Type II. polypropylene / polycarbonate, Lignocel3/4-FASERN Hygienic Animal Bedding available
- Diet (e.g. ad libitum): ssniff SM Rat/Mouse – “Breeding & Maintenance", ad libitum
- Water (e.g. ad libitum): tap water from the municipal supply from 500 mL bottle, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-26.8
- Humidity (%): 33-79
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

50% solution in DMF

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: soluble in DMF at a maximum of 50% (w/v), forming a brown transparent solution. Doses of 50%, 25% and 10% (w/v) were used in the preliminary study.
- Irritation: All animals scratched at the dosage sites on day 1, and alopecia was observed at the 50% dose group on d 2-6 and in the 25% dose group on days 3-6. No significant increase in ear thickness was seen, nor was there any indication of irritation at the site of application.
- Lymph node proliferation response: the draining lymph nodes were visually examinate dan the appearance was normal in all animals.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

The Local Lymph Node Assay is considered valid if it meets the following criteria:
- the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
- the positive control substance produces a significant lymphoproliferative response increases (SI>3),
- each treated and control group includes at least 4 animals,
- the test item does not cause serious systemic or local toxicity.



TREATMENT PREPARATION AND ADMINISTRATION:
Animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). Each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed at least twice a day on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
Both ears of each mouse were observed for erythema and ear thickness was measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
On Day 6, animals were intravenously injected via the tail vein with 250 µL of sterile PBS containing approximately 20 µCi of 3HTdR. Once injected, the mice were left for 5 hours (± 30 minutes). The mice were euthanized by asphyxiation with ascending doses of carbon dioxide. The draining auricular lymph nodes were excised and the nodes of mice from each test group were pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS. A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregation. Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 degrees C. Pellets were gently resuspended and
washed twice. The pellets were finally resuspended in 3 mL of 5% (w/v) TCA solution. After overnight incubation at 2-8 degrees C, precipitates were centrifuged and pellets were resuspended in TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement) of decays per minute. Background DPM level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Positive control substance(s):

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

Statistics:

DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.
The EC3 value of the test item (= concentration of test item which results in a SI of 3) was calculated. The calculation of the EC3 value was conducted by linear interpolation according to the equation: EC3 = c + [(3-d)/(b-d)] x (a-c)
where the data points lying immediately above and below the SI value of 3 on the LLNA dose-response plot have the co-ordinates (a,b) and (c,d) respectively.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation indices for the test material at 50%, 25%, 10% and 2% (w/v) dilution were 24.4, 16.8, 13.3 and 2.9.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Net DPMs in the vehicle control, 50%, 25%, and 10% were 3259.5, 79656.5, 54843.5, 43335.5, respectively. In a second experiment, the vehicle control was 240.9 and the 2% test item was 695.6 DPM.

Any other information on results incl. tables

No mortality or signs of systemic toxicity were observed during the study. There was no significant change in body weights between the dose groups. A minimal amount of test item precipitate was observed on the ears of the animals in the 50% (w/v) dose group on Day 2. All animals scratched the application side on day 1, and some alopecia was observed on day 2 (50% group) and day 3 (25% group).

DPM, DPN and Stimulation Index Values for all Groups (Experiment.1)

Test GroupName	Measured DPM /group	DPM	Number of lymphnodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	74 31	-	-	-	-
Negative (vehicle) control( DMF)	3312	3259.5	8	407.4	1.0
ZAN573 (50% (w/v) in DMF)	79709	79656.5	8	9957.1	24.4
ZAN573 (25% (w/v) in DMF)	54896	54843.5	8	6855.4	16.8
ZAN573 (10% (w/v) in DMF)	43388	43335.5	8	5416.9	13.3
Positive control (25% (w/v) HCA inDMF)	21385	21332.5	8	2666.6	6.5

DPM, DPN and Stimulation Index Values for Additional Dose Group (Experiment 2)

Test Group Name	Measured DPM/ group	DPM	Numberoflymphnodes	DPN	Mean DPM/group	Stimulation Index
Background(5% (w/v)TCA)	31 32	-	-	-	-	-
Negative (vehicle) control (DMF)	477	445.5	2	222.8	240.9	1.0
	462	430.5	2	215.3
	687	655.5	2	327.8
	636	604.5	2	302.3
	304	272.5	2	136.3
ZAN573 (2% (w/v) in DMF)	5599	5567.5	8	695.9	695.9	2.9
Positive control (25% (w/v) HCA in DMF)	2061	2029.5	2	1014.8	1506.6	6.3
	1654	1622.5	2	811.3
	4185	4153.5	2	2076.8
	2921	2889.5	2	1444.8
	4402	4370.5	2	2185.3

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information meets classification criteria for 1B or 1A (residual) Criteria used for interpretation of results: EU

Conclusions:

In an LLNA study, ZAN 573 shows a Stimulation Index above 3, and so is concluded to be skin sensitiser. The calculated EC3 value is 2.1% (w/v). Based on this value, the test item is classified as skin sensitizer Category 1 (sub-category 1B). The presence of trimellitic anhydride as a residual indicates the substance should be classified as sub-category 1A. This study is informative for evaluation of the toxicity of members of the cyclic acid anhydride chemical category, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.