AAA reaction product

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-04-30 to 2003-05-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed in accordance to guideline with no deviations

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): DEA/ACID ANHYDRIDE REACTION PRODUCT

- Physical state: highly viscous amber liquid

- Composition of test material, percentage of components:
Monomers: <17-35% (9-16% diethanolamine; 2-8% tetrahydrophthalic acid; 5-11% trimellitic acid; <0.1% tetrahydrophthalic anhydride; <0.8% trimellitic anhydride)
Dimers/trimers: <24-45%
Polymers: <10-25%
Not removable water: 5-15%

- Lot/batch No.: 200501.UN2810

- Expiration date of the lot/batch: september 2003

- Stability under test conditions: stable under storage condition

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: mouse, CBA/CaOlaHsd
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization)
- Weight at study initiation: 16.9 g - 21.0 g (beginning of acclimatization period)
- Housing: In groups of five in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH 4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 84/02 mouse maintenance diet available ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen, available ad libitum.
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

IN-LIFE DATES: From 30-APR-2003 to 14-MAY-2003

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Test groups: 5 %, 10 % and 25 % AAA reaction product (w/w) in N,N-Dimethylformamide .
Positive control group: 25 % (w/w) ALPHA-HEXYLCINNAMALDEHYDE in N,N-Dimethylformamide.
Negative control group: N,N-Dimethylformamide

No. of animals per dose:

Number of animals in test group: three groups each of 5 female mice - a total of 15
Number of animals in negative control group: 5 female mice
Number of animals in positive control group: 5 female mice

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in 2 mice with concentrations of 1 %, 5 %, 10 % and 25 % (w/w) (pretest excluded from Statement of Compliance).

- Irritation: No irritation effects were observed at these concentrations after a single application. The test item in the main study was assayed at three consecutive concentrations. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

- Lymph node proliferation response: not determined in the range finding test

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled. First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (S.I.). Second, that 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.


TREATMENT PREPARATION AND ADMINISTRATION:

Positive control substance(s):

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

Statistics:

The mean values and standard deviations of body weights and DPM values were calculated. T-test and Dunnett-test were conducted for assessment of the difference significance between the test item groups, positive control group and the negative control (vehicle) group.

Results and discussion

Positive control results:

Stimulation index of 7.7. was obtained for the positive control substance ( Alpha hexyl-cinnamaldehyde in N,N-Dimethylformamide)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 5%: 1.2 10%: 1.0 25%: 1.2 Positive control: 7.7

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The proliferative responses of lymph node cell is expressed as the number of radioactive disintegrations per minute per mouse (DPM/MOUSE). The test and control group mean DPM per mouse were calculated. The ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX) (S.I.) was calculated by dividing the mean DPM for each test group by the mean DPM of the vehicle group. Before DPM/MOUSE values were determined, mean scintillation-background DPM was subtracted from test and control raw data. 3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 µl of 88.3 µCi/ml 3HTdR (equal to 22.1 µCi 3HTdR) by intravenous injection via a tail vein. The following mean DPM values were obtained: Negative control group: 819±84 Positive control group: 6280±2269 5%: 1024±256 10%: 857±291 25%: 985±152

Any other information on results incl. tables

Summary table.

Group		% (w/w)	DPM/MOUSE M±SD	SI (SD)	Statistical Analysis ^a)t-test (G = 2, N = 10, t = 2.31) ^b)Dunnett-test (G = 4, N = 20, t = 2.59)
					t value	Conclusion
NCG 1		-	819±84	-	-	-
PCG 2		25	6280±2269	7.7 (2.8)	5.38^a)	**
TG 3		5	1024±256	1.2 (0.3)	1.53^b)	--
TG 4		10	857±291	1.0 (0.4)	0.28^b)	--
TG 5		25	985±152	1.2 (0.2)	1.24^b)	--

**	significant difference at p£0.05 (two sides)
--	no significant difference at p£0.05 (two sides)

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The possible contact allergenic potential of AAA reaction product was evaluated in accordance to OECD 429 (LLNA test). Stimulation index of 1.2, 1.0 and 1.2 were determined with AAA reaction product at concentrations of 5 %, 10 % and 25 % (w/w), respectively. For the positive control substance, the stimulation index was 7.7 indicative of a valid study design. Based on these data, AAA reaction product was found to be a non-sensitizer when tested at concentrations of up to 25 % (w/w) in N,N-Dimethylformamide.

Executive summary:

The possible contact allergenic potential of AAA reaction product was evaluated in accordance to OECD 429 (LLNA test).

Three groups each of five female mice were treated daily with AAA reaction product at concentrations of 5 %, 10 % and 25 % (w/w) in N,N-Dimethylformamide by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A positive control group of five mice was treated with 25 % (w/w) Alpha-hexylcinnamaldehyde in N,N-Dimethylformamide. A negative control group of five mice was treated with the vehicle (N,N-Dimethylformamide) only.

Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per mouse. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes.

No test item-related clinical signs were observed in any animals of negative control group, Group 3 (TI at 5 %) or Group 4 (TI at 10 %). At the observation of two hours after the first application, a slight ear swelling was observed at both dosing sites in all mice of the positive control group. On the third application day and one day after, the swelling increased to moderate and slight ear erythema at both dosing sites was also noted. The ear swelling persisted for the remainder of the in-life phase of the study. On the third application day, a slight ear swelling was observed at both dosing sites in all mice of Group 4 (TI at 25 %), persisting for two days. Three days after the first application, one animal (No. 25) showed hair loss, persisting for the remainder of the in-life phase of the study.

In this study, stimulation index of 1.2, 1.0 and 1.2 were determined with AAA reaction product at concentrations of 5 %, 10 % and 25 % (w/w), respectively. For the positive control substance, the stimulation index was 7.7 indicative of a valid study design. Based on these data, AAA reaction product was found to be a non-sensitizer when tested at concentrations of up to 25 % (w/w) in N,N-Dimethylformamide.