Resin acids and Rosin acids, reaction products with formaldehyde, calcium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 November 2012 to 02 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

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:

Resin acids and Rosin acids, reaction products with formaldehyde, calcium salts

EC Number:

696-130-6

Cas Number:

1364681-45-7

Molecular formula:

Unspecified

IUPAC Name:

Resin acids and Rosin acids, reaction products with formaldehyde, calcium salts

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Resin acids and rosin acids, reaction products with formaldehyde, calcium salts
- Physical state: Solid, offwhite
- Analytical purity: 96.0 % w/w
- Lot/batch No.: DMG 7245
- Expiration date of the lot/batch: July 05, 2016
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature
- Homogeneity: The test substance was homogeneous by visual inspection.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Animal strain: Mouse / CBA/CaOlaHsd
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8 - 12 weeks
- Mean weight at study initiation: ca. 18 - 23 g
- Housing: Single housing; Makrolon cage, type II
- Diet: STANRAB (P) SQC; SDS Special Diets Services, Altrip, Germany
- Water: Tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30- 70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From 09 November 2012 to 19 November 2012

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

(MEK)

Concentration:

5%, 2% and 1% (w/w) in the vehicle MEK.

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was 25% (w/w). The dilutions were formulated in methyl ethyl ketone (MEK).
- Irritation: To determine the highest non-irritant test concentration, two pre-tests (experimental conduct in accordance with GLP but without a GLP status) were performed. Two mice per concentration were treated epicutaneously with test item concentrations of 2.5, 5, 10 and 25% (w/w) each on three consecutive days. Clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an analytical balance. In addition scaling and white test item residues were noted in both dose groups, while incrustations were only noted in dose group 25%. Animals in dose group 2.5 and 5% did not show increased ear weights or ear swellings > 25%. Scaling was observed in both dose groups prior sacrifice.
- Lymph node proliferation response: Additionally the weight of the pooled lymph nodes from both sides was determined for each animal. Signs of systemic toxicity were not observed in the pre-tests. At the tested concentrations of 10 and 25% the animals showed signs of local irritation as confirmed by the ear weight measurements or ear thickness measurements, respectively. At least one of two animals in dose group 25% showed both increased ear weights > 25% and ear swellings > 25%. One animal in dose group 10% showed also ear swellings > 25%.
The highest concentration tested was the highest level that was achieved whilst avoiding systemic toxicity and excessive local irritation in the pre-test.

MAIN STUDY
TREATMENT SCHEME
Control group 1 Vehicle control in MEK
Test group 2 Test item 1% in MEK
Test group 3 Test item 2% in MEK
Test group 4 Test item 5% in MEK
Test group 5 Positive control 25% HCA in MEK

TREATMENT PREPARATION:
- Daily test item preparation and homogenization until end of each application period: The test item preparation will be produced for each application group shortly before application by stirring with a high speed homogenizer (Ultra-Turrax) and a magnetic stirrer.
- Test item homogenization until end of each application period: The homogeneity of the test item during application will be ensured by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the test item was suspended in the vehicle.

VEHICLE
- Vehicle: MEK (methyl ethyl ketone)
- Reason for the selection of the vehicle: MEK was used as the vehicle because good homogeneity of the preparation was achieved.
- Form of application: Suspension

EXPERIMENTAL PROCEDURE
- Route of application: Epicutaneously to the dorsum of both ears
- Application volume: 25 μL per ear
- Site of application: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- BrdU injection: On study day 4 mice were injected inter-peritoneally (i.p.) with 0.5 mL of 10 mg/mL BrdU in sterile phosphate-buffered physiological saline.
- Determination of ear weight: Immediately after sacrifice a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal using an analytical balance. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: Subsequently, the auricular lymph nodes (right and left) were dissected and total weight of both lymph nodes of each animal was determined.
- Preparation of cell suspension and determination of cell count: All lymph nodes (per test group) were carefully passed through a cell strainer and immediately afterwards, the cell suspension was diluted further with an isotone buffer solution and measured with a cell counter straight away.
- Measurement of BrdU in the lymph node cells: BrdU is measured by Elisa using a commercial kit (Roche Applied Science, Mannheim, Germany)

- Evaluation of results
The cell proliferation induced in the draining lymph node (auricular lymph node) after epicutaneous administration was used for evaluating the skin sensitizing potential. BrdU incorporation into the lymph node cells and the cell count, which was determined after cell isolation from both ear lymph nodes, indicates proliferation. Since certain skin irritants also induce cell proliferation and swelling of the draining lymph nodes of the application site, leading to false positive findings, ear weight as well as the lymph node weight was additionally determined. Especially the ear weight, which is a surrogate for ear swelling, was used as an indicator of irritation.
The effects of test item treatment on BrdU incorporation, cell count of lymph node cells, weight of the lymph nodes as well as ear weight in the test groups was assessed in comparison to the vehicle control group.

CLINICAL EXAMINATIONS
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites was checked individually.
- Mortality: A check for moribund and dead animals was made at least once each workday.

CLINICAL PATHOLOGY
- No examinations were performed.

PATHOLOGY
- The animals were sacrificed on study day 5 about 24 hours after BrdU injection by cervical dislocation. Necropsies were not be performed.
- Animals, which died spontaneously during the observation period, were necropsied as soon as they are found dead and any abnormalities was recorded.

CALCULATION OF RESULTS
- Results for each treatment group are expressed as the mean SI. The SI is derived by dividing the mean BrdU labelling index/mouse within each test substance group and the PC group by the mean BrdU labelling index for the NC group. The average SI for the NCs is then one.
The BrdU labelling index is defined as: BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
Where; em = emission wavelength; and ref = reference wavelength.
- A result is regareded as positive when SI ≥ 1.6.

Positive control substance(s):

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

Statistics:

Calculation of mean and standard deviation was performed.

Results and discussion

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: EC1.6 = 4.36%

Any other information on results incl. tables

Table 1 Stimulation Index. BrdU labeling Index

Test item concentration % (w/w)	Group Calculation
	Mean BrdU labeling index^a)	SD^b)	S.I.
Vehicle (MEK)	0.039	0.0086	1.0
1 % (test substance)	0.055	0.0247	1.4
2 % (test substance)	0.054	0.0159	1.4
5 % (test substance)	0.112	0.0298	2.8
Positive control (HCA)	0.296	0.0378	7.6

a) Mean BrdU labeling index was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals per group)

b) SD = Standard Deviation

S.I. = Stimulation Index (values of the test item groups related to the mean value of the control group)

 

Table 2 Stimulation Index. Lymph Node Weight

Test item concentration % (w/w)	Group Calculation
	Mean Lymph Node Weight (mg)^a)	SD^b)	S.I.
Vehicle (MEK)	4.5	0.3	1.0
1 % (test substance)	5.5	1.0	1.2
2 % (test substance)	5.5	0.3	1.2
5 % (test substance)	7.0	0.2	1.5
Positive control (HCA)	10.7	0.8	2.4

a) Mean Lymph Node Weight was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals per group)

b) SD = Standard Deviation

S.I. = Stimulation Index (values of the test item groups related to the mean value of the control group)

 

 

 

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information