Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 19 February 1991 and 28 March 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Buehler, E. V., Arch Dermatol, 91:171, 1965
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test

Test material

Constituent 1
Chemical structure
Reference substance name:
Sulfonic acids, petroleum, calcium salts
EC Number:
263-093-9
EC Name:
Sulfonic acids, petroleum, calcium salts
Cas Number:
61789-86-4
Molecular formula:
Cannot be adequately determined for this UVCB substance
IUPAC Name:
Petroleum mixture of C10 to 47 saturated linear alkaryl derivatised cyclopentan cyclohexanbenzene sulfonic acid and C10 to 47 saturated linear alkaryl derivatised cyclopentan phenobenzene sulfonic acid calcium salts
Details on test material:
Identification: (Total Base Number = 300)
Date Received: February 5, 1991
Purity: Test material purity data are the responsibility of the Sponsor
Stability: Stable under normal storage conditions
Physical Description: Light brown semi-solid
Storage Conditions: Sealed container at room temperature.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Source: Murphy Breeding Laboratories, Inc., Plainfield, Indiana
Number: Fifteen males and fifteen females on the main study and four males and four females on the Primary Irritation Phase

Bodyweight range: 322 to 395 grams on study day -1 (excluding Primary Irritation Phase animals)

Age at Start of Study: Young adult

Method of Identification: Cage label

Housing: Individual suspended wire-mesh cages. The animals were maintained by the animal husbandry staff of WIL Research Laboratories, Inc. in accordance with standard operating procedures.
Quarantine: The animals used on the main study were acclimated to laboratory conditions for a minimum of 1 days prior to study initiation.
Food and Water: Purina Certified Guinea Pig Chow· #5026 (ad libitum) and tap water from on-site wells (ad libitum). Water is analyzed twice yearly in accordance with S.O.P. No A-020. Results of analyses are available upon Sponsor request. It should be noted that beginning on February 24, 1991,
Purina Certified Guinea Pig Chow· #5026 could not be obtained from the supplier and Purina-Guinea Pig Chow #5025 was fed ad libitum until March 14, 1991. There are no contaminants known which could be expected to interfere with the study. Therefore, this deviation has no effect on the scientific validity, integrity or objective of the study.
Environmental Conditions: Animal room with controlled temperature (69-76°F), humidity (30-69%) and light (12 hours light/12 hours dark).

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
No. of animals per dose:
The Primary Irritation Phase utilized eight guinea pigs. The main study, which included induction, challenge and rechallenge utilized a Test Group of twelve guinea pigs and a Naive Control I and II Group of six guinea pigs in each. In addition, the main study included a Positive Control Group of six guinea pigs.
Details on study design:
The study consists of four phases.
The Primary Irritation Phase consisted of single applications of multiple concentrations of the test material to determine irritation thresholds.
The Induction Phase consisted of multiple applications of test material to stimuIate the immune system.
The Challenge Phase consisted of single applications of the maximal nonirritating concentration of test material to determiine if delayed contact hypersensitivity had occurred.
The Rechallenge Phase consisted of single applications of the maximal nonirritating concentration of the test material to confirm challenge results.
The Primary Irritation Phase utilized eight guinea pigs and evaluated five different concentrations of test material and the vehicle for selection of the appropriate induction and challenge concentrations. The main study, which included induction, challenge and rechallenge utilized a Test Group of twelve guinea pigs that was induced with multiple applications of test material and a Naive Control I and II Group of six guinea pigs in each that were dosed only at the Challenge and RechalIenge phases, respectively. In addition, the main study included a Positive Control Group of six guinea pigs. The Positive Control Group was induced and challenged on an identical regimen as the Test Group and employed a known dermal sensitizer to prove the reliability of the test system.
Animals used on the Primary Irritation Phase were arbitrarily selected from available stock. Animals used on the main study were selected from available stock and assigned to groups by straight unstratified randomization through use of the WIL Research Laboratories, Inc. Computer Data Management System.

Route and Rationale of Test Material Administration:
Direct topical occluded application to shaved intact skin. This route of administration is standard for assessment of the potential to induce delayed contact hypersensitivity by the modIfied Buehler method. The modified Buehler method if an accepted procedure for evaluation of potential for immunologically mediated dermal sensitization.
Test Material Administration:
The guinea pigs' backs were clipped with an electric clipper on the day prior to each dosing. The prepared test and positive control material solutions were maintained on a magnetic stir plate during dosing.
Primary Irritation Phase:
The diluted tesf materials were administered at 0.4 ml/site for weight to weight concentrations in light, white mineral oil and at 0.4 g/site for 100% concentrations. The vehicle, light white mineral oil was administered at 0.4 ml/site. Doses were applied under 25 mm Hilltop Chambers that were occluded with plastic wrap and overwrapped with 75 mm Elastopiast Tape. There were three test sites per guinea pig. The period of exposure was six hours, after which the bandages were removed and wiped with disposable paper towels moistened with light, white mineral oil.
Induction Phase:
Undiluted (100%) test material, and the prepared positive control material, (0.25% w/v DNCB in 80% ethanol) were applied at 0.4g/site and 0.4 ml/site, respectively, to the appropriate animals. Doses were applied under 25 mm Hilltop Chambers, occluded with plastic wrap and overwrapped with 75 mm Elastoplast Tape.
Induction doses were applied to the same site on the left flank of all Test and Positive Control Group animals except that four sites in the Positive Control Group were moved anterior to the prevIous sites for the third induction dose. Test and Positive Control Group animals each received three induction doses spaced one week apart over a period of three weeks. All induction exposure were six hours, after which the bandages were removed. In the Test Group, the test materIal was removed using disposable paper towels moistened with light, white mineral oil. Positive Control Group animals were washed with disposable paper towels moistened with tepid tap water.
All naive control animals remained untreated during the Induction Phase.
Challenge Phase:
Fourteen days after the final induction dose, the test and positive control materials were adininistered to a previously unexposed site on the right anterior flank of the appropriate animals. Doses were applied under 25 mm Hilltop Chambers that were occluded with plastic wrap and overwrapped with 75 mm,Elastoplast Tape. The exposure period was six hours after which the bandages were removed and the sites washed with disposable, paper towels moistened with light, white mineral oil for the Test and Naive Control l Groups and tepid tap water for the Positive Control Group.
Test and Naive Control I Group animals were dosed at 0.4 ml/site with 25% w/w test material in light, white mineral oil. Positive Control Group animals were dosed with 0.1 % w/v dinitrochiorobenzene in 80% ethanol at 0.4 m/site.
Rechallenge Phase:
One week after, the initial challenge, the test material was administered to a previously unexposed site on the right posterior flank of the Test Group and Naive Control II Group animals. Doses were applied under 25 mm Hilltop Chambers that were occluded with plastic wrap and overwrapped with 75 mm EIastoplast Tape. The exposure period was six hours after which ithe bandages were removed and the sites washed with disposable paper towels moistened with light, white mineral oil. Test and Naive Control II Group animals were dosed at 0.4 ml/site with 25% w/w test material in light, white mineral oil.

Study design: in vivo (LLNA)

Positive control substance(s):
other: dinitrochlorobenzene

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% w/w test material in light, white mineral oil
No. with + reactions:
4
Total no. in group:
12
Clinical observations:
No clinical findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% w/w test material in light, white mineral oil. No with. + reactions: 4.0. Total no. in groups: 12.0. Clinical observations: No clinical findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% w/w test material in light, white mineral oil
No. with + reactions:
2
Total no. in group:
12
Clinical observations:
No clinical findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% w/w test material in light, white mineral oil. No with. + reactions: 2.0. Total no. in groups: 12.0. Clinical observations: No clinical findings.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25% w/w test material in light, white mineral oil
No. with + reactions:
4
Total no. in group:
12
Clinical observations:
No clinical findings
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 25% w/w test material in light, white mineral oil. No with. + reactions: 4.0. Total no. in groups: 12.0. Clinical observations: No clinical findings.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25% w/w test material in light, white mineral oil
No. with + reactions:
2
Total no. in group:
12
Clinical observations:
No clinical findings
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 25% w/w test material in light, white mineral oil. No with. + reactions: 2.0. Total no. in groups: 12.0. Clinical observations: No clinical findings.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% w/w test material in light, white mineral oil
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
No clinical findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% w/w test material in light, white mineral oil. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: No clinical findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% w/w test material in light, white mineral oil
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
No clinical findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% w/w test material in light, white mineral oil. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: No clinical findings.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
25%w/w test material in light, white mineral oil.
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
No clinical findings
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%w/w test material in light, white mineral oil.. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: No clinical findings.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
25% w/w test material in light, white mineral oil.
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
No clinical findings
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% w/w test material in light, white mineral oil.. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: No clinical findings.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1% w/w concentration of dinitrochlorobenzene in 80% ethanol.
No. with + reactions:
6
Total no. in group:
6
Clinical observations:
No clinical findings
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1% w/w concentration of dinitrochlorobenzene in 80% ethanol.. No with. + reactions: 6.0. Total no. in groups: 6.0. Clinical observations: No clinical findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1% w/w concentration of dinitrochlorobenzene in a 80% ethanol.
No. with + reactions:
6
Total no. in group:
6
Clinical observations:
No clinical findings
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.1% w/w concentration of dinitrochlorobenzene in a 80% ethanol.. No with. + reactions: 6.0. Total no. in groups: 6.0. Clinical observations: No clinical findings.

Any other information on results incl. tables

Naive Control Group animals 02003169 and 02003275 had wet or dry yellow urogenital staining at study termination. There,were no other clinical findings during the study. Body Weights: There were no remarkable changes or differences observed in bodyweights. Dermal Observations: Induction Phase (Table 6 - attachment 1): The 100% test material induced eight and six grade ± reactions at the 24 -hour and 48 - hour readings, respectively, following the first induction dose. After the second induction dose there were seven grade ± reactions at the 24 and 48 hour readings. Five and three grade ± reactions were noted at the 24 and 48 hour readings, respectively, after the third induction dose. Five grade ± reactions were noted on positive control animals after the first induction dose. One grade ± and five grade 1 reactions were noted on positive control animals after the second induction dose. One animal had eschar and necrosis after the second induction dose. Following the third induction dose there were one grade ± four grade 1 and one grade 1 reactions. Two animals had necrosis after the third induction dose. Challenge Phase (Tables 7 and 8 - attachment 2 and 3): There were eight grade ± and four grade 1 reactions at 24 hours for Test Group guinea pigs challenged with 25% w/w test material in light, white mineral oil. Ten grade ± and two grade 1 reactions were noted at 48 hours. There were four very slight (grade ±) reactions at 24 hours for Naive Control Group I guinea pigs challenged with 25% w/w test material in light, white mineral oil. There were five very slight (grade 1 reactions at 48 hours) Reactions in Positive Control Group guinea pigs challenged with 0.1% w/v dinitrochlorobenzene in 80% ethanol included one slight (grade 1) and five moderate (grade 2) reactions at 24 hours. There were five slight and one moderate reactions at 48 hours. Rechallenge Phase (Tables 9 and 10 - attachments 4 and 5): There were seven grade ± and four grade 1 reactions at 24 hours for Test Group guinea pigs challenged with 25% w/w test material in light, white mineral oil. Nine grade ± and :two grade 1 reactions were noted at 48 hours. There were four very slight (grade 1) reactions at 24 hours for Naive Control Group II guinea pigs challenged with 25% w/w test material in light, white mineral oil. There were six very slight grade 1 reactions at 48 hours. Incidence and Severity Indices: The Sensitization Incidence Indices were calculated to be 4/12 (33%) for the Test Group following challenge dosing. The Irritation Severity Indices were 0.7 and 0.6 at 24 and 48 hours, respectively, for the Test Group. The Irritation Severity Indices were 0.3 and 0.4 at 24 and 48 hours, respectively, for the Naive Control I Group. The Sensitization Incidence Index was calculated to be 6/6 (100%) for the Positive Control Group followirig challenge dosing. The Irritation Severity Indices were 1.8 and 1.2 at 24 and 48 hours, respectively, for the Positive Control Group. The Sensitization Incidence Index was calculated to be 5/12 (42%) for the Test Group following rechailenge dosing. The Irritation Severity Indices were 0.6 and 0.5 at 24 and 48 hours, respectively, for the Test Group. The Irritation Severity Indices were 0.3 and 0.5 at 24 and 48 hours, respectively, for the Naive Control-II Group Ranking of Sensitization Potential: Based on the Sensitization Incidence Indices of 33% and 42% following challenge and rechallenge dosing, respectively, the test material, was found to be a sensitizing agent in the albino guinea pig under the conditions of this study. The positive control material, dinitrochlorobenzene, was demonstrated to be an extreme sensitizing agent under the conditions of this study based on the sensitization incidence index of 100%.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Based on the data obtained, the test material was found to be a sensitizing agent in albino guinea pigs under the conditions of this study
Conclusions:
There were no deaths, test material related clinical findings or body weight changes.
There were four and five sensitization reactions observed in the Test Group that exceeded the highest reactions in the Naive Control Groups following challenge and rechallenge dosing, respectively. The Sensitization Incidence Indices for the Test Group were 33% (initial challenge) and 42% (rechallenge).
The positive control material, dinitrochlorobenzene, was demonstrated to be an extreme sensitizing agent in albino guinea pigs under the conditions or this study based on the Sensitization Incidence Index of 100%, thereby demonstrating the reliability of the test system. Based on the data obtained, the test material was found to be a sensitizing agent in albino guinea pigs under the conditions of this study.
Executive summary:

The sensitization potential of the test material (Total Base Number = 300) was evaluated in this modified Buehler method dermal sensitization study.

A Test Group of six male and six female Hartley albino guinea pigs was dosed topically with the test material one time per week for three weeks for a total of three induction exposures. The duration of the exposures was six hours. Two weeks after the last induction exposure, Test Group animals were challenge dosed for detection of sensitization by topical application of known nonirritating concentration of the test material to previously unexposed areas of skin. One week after challenge dosing, Test Group animals, were rechallenged to confirm the initial challenge results. (The test material concentrations used for induction, challenge and rechallenge dosing were selected based on the resuits of range-finding experimentation in the Primary Irritation Phase.)

A Positive Control Group of three male and three female guinea pigs was included to demonstrate the reliability of the test system. The Positive Control Group was induced and challenged on a similar regime as the Test Group with dinitrochlorobenzene (DNCB) as the positive control material.

Naive Control I and II Groups of three male and three female guinea pigs each were dosed only at challenge and rechallenge, respectively, in the same manner as the Test Group, and served as irritation controls. Reactions to challenge and rechallenge dosing were evaluated at approximately 24 and 48 hours after completion of exposure.

Body weights and clinical observations were recorded just prior to study initiation and at termination.

There were no deaths, test material related clinical findings or body weight changes.

Based on the Sensitization Incidence Index of 100%, the positive control material, DNCB, was found to be an extreme sensitizing agent in the albino guinea pig under the conditions of this study, thereby demonstrating the reliability of the test system.

There were four and five sensitization reactions in the Test Group that exceeded the highest reactions observed in the Naive Control Groups following challenge and rechallenge dosing, respectively. The Sensitization Incidence Indices were calculated to be 33% and 42% for the Test Group following challenge and rechallenge dosing, respectively. Based on these results, the test material was found to be a sensitizing agent in albino guinea pigs under the conditions of this study.