Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Acetonitrile (HPLC Grade)
- Physical state: Clear colorless liquid
- Analytical purity: 99.9+%, HPLC Grade
- Lot/batch No.: ER0475ILQ
- Storage condition of test material: Room temperature under a blanket of Nitrogen. Test material was stored in the original container.
- Supplier: Aldridge Chemical Company

Positive Control: alpha Hexylcinnamaldehyde, tech., 85%
- Lot Number: 10021HF
- Physical Description: A clear, yellow liquid
- Storage Conditions: Room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Cage cards incorporating animal group code-arrival date-color code marking system were used to identify each guinea pig.

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. P.O. Box 29176 Indianapolis, IN 46229
- Age at study initiation: Young adult
- Weight at study initiation: At the start of the pilot phase of testing the pilot animals weighed 458 to 594 grams. At the start of the induction phase of testing the test, distilled water control, positive control, and naive positive control animals weighed 422 to 703 grams.
- Housing: The animals were randomly caged according to Standard Operating Procedures and individually housed- in wire mesh suspension cages.
- Diet: Teklad Guinea Pig Diet
- Water: ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature: 64-79°F
- Humidity: 30-70%
- Photoperiod: 12-hour light/12-hour dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
undiluted
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
undiluted
No. of animals per dose:
A total of 53 animals were utilized. Twenty test animals, ten distilled water control animals, ten positive control animals, five naive positive control animals, and eight pilot animals were used. Equal numbers of males and females were included in each animal group, as possible.
Details on study design:
RANGE FINDING TESTS: The irritation phase had the purpose of determining the proper level of test material to be used in the induction and challenge phases. The irritation potential of the test material at levels of undiluted, 50%, 25%, and 10% was evaluated in four animals. One level of the test material was evaluated per animal. Dilutions of the test material were prepared w/v in distilled water. The day prior to test material exposure, the hair was removed from each of the animal's backs using a small animal clipper. Closed patches were applied to the animals in the following manner: A 0.3 ml quantity of each test preparation was applied into a 25 mm Hill Top Chamber®. The animal was placed into the restrainer, and the chambers were applied to the clipped surface as quickly as possible. The chambers were occluded with rubber dental dam pulled taut and fastened to the bottom of the restrainer with clips. The restrainer was adjusted to minimize movement of the animal during exposure. Approximately six hours later, the dental dam and chambers were removed and the animal was taken from the restrainer and placed in its cage.

A second pilot was conducted to evaluate the irritation potential of the undiluted test material. A single patch was applied to four additional pilot animals utilizing the same exposure procedure as for the "Irritation Screening Phase".

MAIN STUDY
A. INDUCTION EXPOSURE
The left shoulder (Site 1) of each test, distilled water control, and positive control animal was clipped with a small animal clipper the day before exposure. The animals were restrained, and a 0.3 ml quantity of the undiluted test material was applied to the test animals. Undiluted distilled water was applied to the distilled water control animals. The positive control material was applied to the positive control animals as a 2.5% w/v formulation in 95% ethanol. The appropriate material was applied using a Hill Top Chamber®. The procedure was repeated at the same site once a week for the next two weeks for a total of three approximate six-hour exposures (the interval between induction exposures varied from 6 to 7 days). After the last induction exposure, the animals were left untreated for approximately two weeks (13 days) before primary challenge.

B. CHALLENGE EXPOSURE
The test, distilled water control, and positive control animals, which had three previous exposures to the appropriate test material at appropriate intervals, were again exposed in the challenge phase approximately two weeks after the last induction exposure. In addition, five naive positive control animals, which had never been exposed to the positive control material, were concurrently treated with the same concentrations.

The same exposure procedure as for the "Induction Phase" was used except the chambers were applied to a skin site that had not been exposed previously. Each test animal received one patch of the undiluted test material using Site 2. Each distilled water control animal received one patch of the undiluted test material and one patch of distilled water material using Sites 2 and 5. Each positive and naive positive control animal received the positive control material at concentrations of 5%, 2.5%, and 1% formulated w/v in acetone using Sites 2, 4, and 5. The patch site was rotated for possible site-to-site variation in response.
Challenge controls:
Yes
Positive control substance(s):
yes

Study design: in vivo (LLNA)

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data were not analyzed statistically.

Results and discussion

Positive control results:
For validation of the test system, a positive control group was evaluated concurrently with the test group. Following primary challenge using alpha Hexylcinnamaldehyde, tech., 85% as 5%, 2.5%, and 1% w/v formulations in acetone, the incidence of grade 1 responses or greater in the test group (10 of 10), (9 of 10), and (6 of 10), respectively was compared to that of the naive positive control group (0 of 5), (0 of 5), and (0 of 5), respectively. The incidence and severity of these responses in the positive control group were significantly greater than those of the naive positive control group indicating that sensitization had been induced at all levels tested.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Undiluted acetonitrile
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
slight, patchy erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Undiluted acetonitrile. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: slight, patchy erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Undiluted Acetonitrile
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
slight, patchy erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Undiluted Acetonitrile. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: slight, patchy erythema.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Undiluted Acetonitrile
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
slight, patchy erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Undiluted Acetonitrile. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: slight, patchy erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Undiluted Acetonitrile
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
slight, patchy erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Undiluted Acetonitrile. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: slight, patchy erythema.
Reading:
1st reading
Hours after challenge:
24
Group:
other: distilled water
Dose level:
undiluted
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
slight patchy erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: distilled water. Dose level: undiluted. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: slight patchy erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: distilled water
Dose level:
undiluted
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
slight patchy erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: distilled water. Dose level: undiluted. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: slight patchy erythema.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
(5) slight but confluent, or moderate patchy erythema; (5) moderate erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 1%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: (5) slight but confluent, or moderate patchy erythema; (5) moderate erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
1%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
(4) slight but confluent, or moderate patchy erythema; (6) moderate erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 1%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: (4) slight but confluent, or moderate patchy erythema; (6) moderate erythema.
Reading:
1st reading
Hours after challenge:
24
Group:
other: naive positive control
Dose level:
1%
No. with + reactions:
3
Total no. in group:
5
Clinical observations:
(3) slight, patchy erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: naive positive control. Dose level: 1%. No with. + reactions: 3.0. Total no. in groups: 5.0. Clinical observations: (3) slight, patchy erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: naive positive control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: naive positive control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Following primary challenge using undiluted Acetonitrile (HPLC Grade) there were no grade 1 responses noted in any animals. The incidence of grade ± responses in the test group (3 of 20) was compared to that of the distilled water control group (2 of 10). The incidence and severity of these responses in the test group were essentially comparable to those produced by the distilled water control group indicating that sensitization had not been induced.

For validation of the test system, a positive control group was evaluated concurrently with the test group. Following primary challenge using ct - Hexylcinnamaldehyde, tech., 85% as 5%, 2.5%, and 1% w/v formulations in acetone, the incidence of grade 1 responses or greater in the test group (10 of 10), (9 of 10), and (6 of 10), respectively was compared to that of the naive positive control group (0 of 5), (0 of 5), and (0 of 5), respectively. The incidence and severity of these responses in the positive control group were significantly greater than those of the naive positive control group indicating that sensitization had been induced at all levels tested.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In conclusion, under the conditions of this test, Acetonitrile (HPLC Grade) was found to be non-irritating and non-sensitizing to guinea pig skin.
Executive summary:

In a guideline (OECD 406) and GLP study, Acetonitrile (HPLC Grade) was found to be non-irritating and non-sensitizing to guinea pig skin when tested by the Buehler method.