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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is GLP and follows the OECD guideline 429 although there is no information on analysis of the test material, dosing and progress inspections.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(1) No in-progress inspection (2) No analysis of test material and dosing solutions
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Details on test material:
CAS #98-94-2, Air Products and Chemicals, Lot #AW4 10736M4

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
Female BALB/c mice, supplied by Charles River, were used throughout this study. The mice were ordered, as needed, and the age of the mice ranged from 9 to 11 weeks. The mice were identified by marking the tails with permanent ink applied from felt-tip pens. A system of different colored inks, hash marks on the tail and, either underlining the number or marking an additional hash mark on the tip of the tail, was used to ensure that each mouse in the test was uniquely identified. The same color of tape, which contained additional information as necessary to maintain the unique identity, was placed over the appropriate animal cage.The animals were housed singly in wire bottom cages suspended above catch pans containing absorbent cageboard. The cages were sanitized and cageboards changed regularly in accordance with good husbandry practices. The cages contained stainless steel feeders filled with Purina Certified Rodent Chow #5002 and a pressure-activated, stainless steel water nipple attached to an automatic water line. The testing facility has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The animal rooms of the testing facility are designed to maintain adequate environmental conditions concerning temperature, humidity, and photocycle and are regulated for the species under test. The temperature is set at 22 ± 1°C, the humidity is set at 50 ± 10% RH and the light/dark cycle is set at 12 hours on: 12 hours off. Alarms are set to sound if the temperature is lower than 19 °C or higher than 25 °C and/or the relative humidity is lower than 39% or higher than 71 %. These limits are based on AAALAC recommendations.Feed analysis is performed by Purina Mills, Inc. to confirm that the diet provides adequate nutrition and to quantify the levels of selected contaminants associated with the formulation process. Analysis of the tap water (municipal water supply) is performed in accordance with Laboratory Standard Operating Procedures.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The amine catalyst was tested in the LLNA at the minimal irritating concentration (MIC). The MIC was determined in previous Primary Dermal Irritation Study (PDIS) as the effective concentration causing a 10% ear swelling (EC10). CAS #98-94-2 was tested at 30% in AOO in the LLNA test.
No. of animals per dose:
5 animals/dose; tested in duplicate
Details on study design:
The Local Lymph Node Assay (LLNA) was used to assess the potential of the test article to cause skin sensitization. The amine catalyst was tested in the LLNA at the minimal irritating concentration (MIC). The MIC was determined in previous Primary Dermal Irritation Study (PDIS) as the effective concentration causing a 10% ear swelling (EC10). This study consisted of two separate trials. Each trial contained a vehicle control group (5 mice treated with a solution of 4:l acetone:olive oil, AOO), three positive control groups (5 mice treated with a solution of each 1.0% dinitrochlorobenzene (DNCB), 0.17% benzalkonium chloride (BC), and 25% trimellitic anhydride (TMA), and test article treatment group (5 mice treated with a 30% solution of the amine catalyst in AOO). The choices of AOO as the vehicle carrier and the three positive controls (DNCB, BC, and TMA) were based on historical data generated in the laboratory. Prior to the application of the solutions on Day 1, an erythema score was assessed for each mouse (grading scale 0 to 4). The solutions were applied by dispensing 12.5 µl of the appropriate solution onto the dorsal and ventral side of each ear using an automatic pipet. Each ear received a total of 25 µl of solution for a total of 50 µl per mouse. The solutions were applied once daily for three consecutive days. On Day 5, prior to injection with 3H-thymidine, the erythema score was assessed for each mouse. Each mouse was then injected, via the lateral tail vein, with 0.2 ml of phosphate buffered saline (PBS) containing 20 µCi of 3H-thymidine. Five hours after the injections, the mice were sacrificed by C02 inhalation and the draining auricular lymph nodes were excised and pooled for each mouse. A single cell suspension of lymph node cells (LNC) was prepared and 3H-thymidine incorporation was measured on a ß-scintillation counter. Radioisotope incorporation was measured as disintegrations per minute (dpm) per mouse and a mean dpm value +/- SE (standard error) was calculated for each experimental group. In addition, a stimulation index (SI) was calculated using absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle control mice as the denominator. A mean SI +/- SE was calculated for each experimental group. Historically, any chemical that produces a SI of > 3 in the LLNA is considered "positive".
Positive control substance(s):
other: 25% trimellitic anhydride (TMA)... (see attached file)
Statistics:
Calculations were made using Microsoft Excel. The following equations were used:1. SI = dpm of mouse mean dpm of vehicle group2. AVERAGE* = "average" of numbers3. STDEV* = "estimates standard deviation of a population based on a sample"4. Standard Error (SE) = STDEV s the square root of the number of mice in group*Defined by Microsoft Excel

Results and discussion

Positive control results:
BC showed a mean erythema score on day 5 of 0, which is consistent with the fact that 0.17% is the MIC. One percent DNCB showed a mean erythema score of 1.0. 25% TMA showed a mean erythema score of 2 on day 5. Both of the sensitiser positive controls produced a robust response with a simulation index (SI) > 70.0. BC triggered a SI which was greater than 3.0 would be considered positive.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.27
Variability:
0.4
Test group / Remarks:
30%
Key result
Parameter:
SI
Value:
2.45
Variability:
0.6
Test group / Remarks:
30%

Any other information on results incl. tables

CAS #98-94-2 was first assayed on November 14, 1996. Erythema was graded based on a grading scale of 0 to 4, with a score of 1 for DCNB and 2 for TMA. There was no irritation (i.e., all erythema scores were 0) at the specified doses for the test article. BC, the irritant positive control, showed a mean erythema score on Day 5 of 0, which is consistent with the fact that 0.17% is the MIC. One percent DNCB showed a mean erythema score of 1.0 and 25% TMA showed a mean erythema score of 2.0 on Day 5. For the results of the LLNA both of the sensitizer positive controls produced a robust response with SI's > 70.0. BC triggered a SI which was greater than 3.0 and would be considered a "positive". The criteria for a SI > 3.0 is based on historical results which recognize that a strong irritant can trigger proliferation in the draining lymph node.  The results of the erythema scores indicate no irritation (i.e., all erythema scores were 0) at the specified dose. BC, the irritant positive control, showed a mean erythema score on Day 5 of 1.8, which is consistent with the fact that 0.17% is the MIC. One percent DNCB showed a mean erythema score of 3.0 and 25% TMA showed a mean erythema score of 3.0 on Day 5. For the results of the LLNA the SI for the positive controls: SI of 3.28 for 0.17% BC, SI of 40.90 for 1.0% DNCB, and SI of 41.07 for 25% TMA are consistent with previous results. Both of the sensitizing positive controls produced robust responses.  BC, the irritant positive control, also produced a SI > 3.0 in these two trials which would trigger a determination that it was "positive" in these two assays. The criteria that a chemical must trigger a SI > 3.0 is based on historical results which recognize that an irritant can trigger a proliferation in the draining lymph node. The SI for the amine catalyst was not greater than 3.0, indicating it is not a sensitizer.

Table 1. Summary of Erythema Results for November 14, 1996:

Group

Concentration

Erythema Score (Day 5)

DCNB

1.0%

1.0

BC

0.17%

0

TMA

25%

2.0

CAS 98-94-2

30%

0

 

Table 2. Summary of LLNA Results for November 14, 1996:

Group

Concentration

SI (Mean +/- SE)

DCNB

1.0%

74.16 +/- 7.20

BC

0.17%

4.77 +/- 1.19

TMA

25%

72.55 +/- 7.65

CAS 98-94-2

30%

1.27 +/- 0.40

 

 

Table 3. Summary of Erythema Results for February 10, 1997:

Group

Concentration

Erythema Score (Day 5)

DCNB

1.0%

3.0

BC

0.17%

1.8

TMA

25%

3.0

CAS 98-94-2

30%

0

 

Table 4. Summary of LLNA Results for February 10, 1997:

Group

Concentration

SI (Mean +/- SE)

DCNB

1.0%

40.90 +/- 3.89

BC

0.17%

3.28 +/- 0.45

TMA

25%

41.07 +/- 5.27

CAS 98-94-2

30%

2.45 +/- 0.62

 

Overall SI Mean for CAS #98-94-2: 1.86 ± 0.40.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
The results from two separate trials indicate that amine catalyst DMCHA did not produce a positive response, and it is concluded that this catalyst is not a skin sensitizer according to the LLNA when tested at minimally irritating concentrations.
Executive summary:

The Local Lymph Node Assay (LLNA) was used to assess the potential of ten amine catalysts as respiratory sensitizers. The amine catalysts were tested in the LLNA at the minimal irritating concentration (MIC). The MIC was determined in previous Primary Dermal Irritation Studies (PDIS) as the effective concentration causing a 10% ear swelling (EC10). For the amine catalysts where an ECl0 could not be calculated, the materials were tested at the highest concentration which did not trigger an increase in ear swelling. The minimal irritating concentration for cyclohexyldimethylamine (DMCHA) was determined to be 30%. The activity of the amine catalysts were compared to three positive controls: 1.0% dinitrochlorobenzene (DNCB), a known dermal sensitizer, benzalkonium chloride (BC), a known irritant (0.17% is the EC 10 for this chemical), and 25% trimellitic anhydride (TMA), a known respiratory sensitizer. In the studies, mice (5 mice per experimental group) were dermally exposed to test materials, positive control material, or vehicle (4:l acetone:olive oil) for three consecutive days. All mice received 12.5 µl of the test material, positive controls, and vehicle on the dorsal and ventral sides of both ears for a total of 50 µl per day. Erythema, an indication of dermal irritation, was assessed for each mouse on Days 1 and 5 of the study. Five days after the initiation of the study, all mice received an intravenous injection, via the lateral tail vein, of 0.2 ml phosphate buffered saline containing 20 µCi of tritiated thymidine (3H -thymidine). Five hours after injection, the mice were sacrificed by CO2inhalation and the draining auricular lymph nodes were excised. A single cell suspension of lymph node cells (LNC) was prepared and 3H-thymidine in corporation was measured on a p-scintillation counter. Radioisotope incorporation was measured as disintegrations per minute (dpm) per mouse and a mean dpm value ± SE (standard error) was calculated for each experimental group. In addition, a stimulation index (SI) was calculated using absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle control mice as the denominator. A mean SI±SE was calculated for each test material from two separate trials (5 test materials were studied in each of four trials).The overall mean SI ± SE for DMCHA is 1.86 ± 0.4 along with MI. The test substance has a simulation index below 3. Therefore, for DMCHA does not warrant any classification according to Directive 67/548/EEC and Regulation (EC) No 1272/2008.