Asphalt, sulfonated, sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-01-14 to 2008-02-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

purity of the test substance was not provided

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Purity of the test substance was not provided.

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from "The Department of Health of the Government of the United Kingdom"

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Asphalt, sulfonated, sodium salt

EC Number:

269-212-0

EC Name:

Asphalt, sulfonated, sodium salt

Cas Number:

68201-32-1

Molecular formula:

UVCB

IUPAC Name:

Asphalt, sulfonated, sodium salt

Details on test material:

- Name of test material (as cited in study report): Sodium sulfonated asphalt
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): N/A
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: active
- Physical state: black solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 12-23 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum (Certified Rat and Mouse Diet)
- Water (e.g. ad libitum): ad libitum (tap)
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (deg. C): The temperature was controlled to remain within a target range of 19-25 deg C. Any occasional deviation from this target was considered not to have affected the purpose or integrity of the study.
- Humidity (%): The relative humidity was controlled to remain within a target range of 30-70 %. Any occasional deviation from this target was considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): ~15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: N/A To: N/A

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

2.5%, 5%, or 10% w/w in DMSO

No. of animals per dose:

- FIve animals

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: DMSO was chosen as the most suitable vehicle for the test substance.
- Irritation: N/A
- Lymph node proliferation response: N/A
-Other: One mouse was treated by daily application of 25 uL of the test substance as a suspension at a concentration of 10 % w/w in DMSO, to the dorsal surface of each ear for 3 consecutive days (Days 1, 2, 3). This was found to be the highest concentration suitable for dosing in the most suitable vehicle. The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
No signs of systemic toxicity were noted.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3HTdR Incorporation
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test substance or positive control substance was regarded as a sensitizer if at least one concentration of the test substance or positive control substance resulted in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test substance failing to produce a threefold or greater increase in 2HTdR incorporation was classified as a "non-sensitizer".


TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was freshly prepared in DMSO. The mice were treated by daily application of 25 uL of the appropriate concentration to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test substance formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. Five days following the first topical application of the test substance (day 6) all mice were injected via the tail vein with 250 uL of phosphate buffered saline (PBS) containing [3H]-methyl-thymidine giving a total of 20 uCi to each mouse.
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes were excised and processed. 1 mL of PBS was added to the lymph nodes.
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish. The lymph node suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL PBS and added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm for 10 min. Pellet was resuspended in 10 mL PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA).
After ~ 18 h incubation at ~ 4 degrees C, the precipitates were recovered by centrifugation at 2100 rpm for 10 min, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by B-scintillation counting. The "PolyQ" vials counting the samples and scintillation fluid were placed in the sample changer of the scintillator and left for ~ 20 min. After ~ 20 min., the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

Positive control substance(s):

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

Statistics:

Data were processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogeneous datasets, Dunnett's Multiple Comparison test was used and for non-homogeneous datasets, Dunnett's T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:

A stimulation index of greater than 3 was recorded for the positive control at a concentration of 15 % v/v in DMSO. The mean dpm/animal was 4877.88 (p < 0.01) for the positive control.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: - A stimulation index of < 3 was recorded for the three concentrations of the test substance (2.5%, 5%, 10% w/w in DMSO).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: - The mean dpm/animal was 1554.00, 1773.71, 1760.30, 2339.69 for the 0, 2.5%, 5%, and 10% w/w in DMSO test groups, respectively.

Results 3

Parameter:

SI

Value:

1.14

Test group / Remarks:

2.5% w/w in DMSO in 5 animals

Remarks on result:

other: Negative

Results 4

Parameter:

SI

Value:

1.13

Test group / Remarks:

5% w/w in DMSO in 5 animals

Remarks on result:

other: Negative

Results 5

Parameter:

SI

Value:

1.51

Test group / Remarks:

10% w/w DMSO in 5 animals

Remarks on result:

other: Negative

Results 6

Parameter:

SI

Value:

3.14

Test group / Remarks:

Positive control substance, 15% v/v in DMSO in 5 animals

Remarks on result:

other: positive

Any other information on results incl. tables

Individual Disintegrations per Minute and Stimulation Indices
Treatment Group	Animal Number	dpm / Animal^a	Mean dpm / animal (SD)	Stimulation Index^b	Result
Vehicle DMSO	1-1	1113.55	1554.00 (± 616.29)	N/A	N/A
	1-2	1508.32
	1-3	2243.28
	1-4	2095.62
	1-5	809.21
Test Substance 2.5% w/w in DMSO	2-1	1962.85	1773.71 (± 489.31)	1.14	Negative
	2-2	1090.28
	2-3	2316.77
	2-4	1467.58
	2-5	2031.06
Test Substance 5% w/w in DMSO	3-1	1259.81	1760.30 (± 618.19)	1.13	Negative
	3-2	1528.48
	3-3	1192.76
	3-4	2235.44
	3-5	2585.00
Test Substance 10% w/w in DMSO	4-1	1299.64	2339.69 (± 834.69)	1.51	Negative
	4-2	3593.71
	4-3	2540.71
	4-4	2187.25
	4-5	2077.15
Positive Control Substance 15% v/v in DMSO	5-1	8695.73	4877.88** (± 2159.72)	3.14	Positive
	5-2	3601.66
	5-3	4180.21
	5-4	3591.15
	5-5	4320.65
^aTotal number of lymph nodes per animal is 2.
^bStimulation Index of 3.0 or greater indicates a positive result.
**Statistically different from the control group p < 0.01.

CLINICAL OBSERVATIONS AND MORTALITY DATA:

- There were no deaths

- No signs of systemic toxicity were noted in the test animals, the vehicle control animals or the positive control animals

BODYWEIGHT:

- One animal in the 10% group presented an insignificant weight loss with no systemic adverse effects.

- Overall bodyweight changes between days 1 and 6 were comparable to those observed in the vehicle control group over the same period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was considered to be a non-sensitizer under the conditions of the test.

Executive summary:

N/A