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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
32 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 406: GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Official Journal of the European Communities, No. L 383A/134, 29 December 1992
Deviations:
yes
Remarks:
Challenge performed 16 days post-induction instead of 14 days
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Challenge performed 16 days post-induction instead of 14 days
Principles of method if other than guideline:
The hazard of allergy in the human population depends on many factors related to usage, such as the concentration of the substance, frequency and duration of exposure to the skin, and whether or not the material will be applied to healthy or inflamed skin, covered or left uncovered, etc. The ideal test method for predicting the sensitization potential of substances would be under conditions which correspond to normal human circumstances (i.e., topical exposure, either with or without occlusion of the treatment site, at concentrations in the range of those to be encountered under foreseeable conditions of use.) However, some materials are very irritating in test animals under conditions of human exposure, and therefore must be applied at a lower concentration in a carrier vehicle; on the other hand, if the material is applied without occlusion, the test procedure becomes rather insensitive, detecting only potent contact allergens. To balance these factors, it has become standard practice to routinely evaluate test materials under occlusive dressings, and to perform whatever dilutions are necessary to avoid any marked irritation or deterioration of the skin at the treatment site.

Numerous procedures for assessing the potential of materials to induce contact sensitization in guinea pigs have been described , among which is that of Buehler. The Buehler guinea pig procedure was chosen because it employs the use of a closed patch system (occluded) which enhances the penetration of topically applied substances, resulting in a much more sensitive method for predicting the sensitization potential of test materials. This procedure has been used for over 30 years and has demonstrated its ability to detect agents which can be strong, moderate, and weak topical sensitizers in humans.
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The Buehler skin sensitisation test is a scientifically valid method for assessing skin sensitisation potential. As the data exists and is adequate for hazard assessment, performing a new LLNA test is not scientifically justified.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
416-740-6
EC Name:
-
Molecular formula:
Can vary from C30H53O4 (di-C11 rxn product) to C36H65O4 (di-C14 rxn product)
IUPAC Name:
Ester reaction products of 1,4-Benzenedicarboxylic acid with C11-14 iso-alcohols, C13-rich
Details on test material:
Test substance is 100% terephthalate ester (Lot # X1274). It is a straw colored liquid.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Fifty-four guinea pigs (27 males and 27 females) of the outbred Hartley albino strain from Charles River Portage (Shaver Road, Portage, Michigan 49081), initially weighing 315.6 g to 412.9 g and approximately one month old, were used.

The animals were housed individually in suspended stainless steel cages and provided with tap water ad libitum via automatic watering devices. Their individual identity was assured by ear tagging and using cage cards. The housing room was set to maintain a constant temperature of 70°F with an acceptable range of 68 to 70°F and the humidistat will be set at 50%, with an acceptable range of 40-60%. (During the performance of this test, the temperature ranged from 68-74°F exceeding the limits of 68-70°F and the relative humidity ranged from 37-70%, exceeding the limits of 40-60%. No noticeable effects on the animals were seen, and it was judged that this excursion did not affect the test). Timing devices were set to provide artificial illumination 12 hours daily. The animals were fed a standard pellet diet (Purina Laboratory Guinea Pig Chow #5025) ad libitum.

The animals were acclimated to the laboratory for at least 12 days before they were placed on study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
Test substance was applied at 75% concentration (w/w).

DNCB positive control was applied at 0.05% concentration in 80% ethanol (w/v, induction phase) or acteone (w/v, challenge phase).
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
Test substance was applied at 75% concentration (w/w).

DNCB positive control was applied at 0.05% concentration in 80% ethanol (w/v, induction phase) or acteone (w/v, challenge phase).
No. of animals per dose:
Twenty guinea pigs exposed to test substance, 10 exposed to DNCB positive control
Details on study design:
Treatment
The test substance animals for this study were randomly assigned to three groups for evaluation of specific parameters: 4 animals for range finding for primary irritation of the test material (Group V), 20 test animals (Group I), and 10 control animals (Group Ill). The control animals were maintained without treatment until primary challenge.

Positive (DNCB) control animals for this study were also randomly assigned to two groups for evaluation of specific parameters: 10 test animals (Group II) and 10 control animals (Group IV).

Primary Irritation
To determine the highest nonirritating concentration of the test material to be used in the challenge phase of the test, a group of four guinea pigs was treated with various concentrations of the test material. Hair was removed from the entire back of 4 guinea pigs using electric clippers. On the following day, four patches of test substance were applied using Hill Top Chambers with 25mm Webril swatches saturated with either 0.4 ml of the test substance (neat), or with the 75.0, 50.0 or 25.0% (w/w) concentrations in mineral oil.

The guinea pigs were wrapped with a piece of rubber dental dam (approximately 5” x 16”) that was placed over the patch sites. The animal was then placed into a restrainer. Another piece of dental dam (approximately 5” x 8”) was draped over the animal’s torso and secured to the sides of the restrainer. After an exposure period of approximately 6 hours, the patches were removed. The treated sites were wiped with saline and cotton gauze to rid the areas of any excess test material. The animals were then returned to their cages.

Approximately twenty-two (22) hours [and not twenty-one (21) hours] after patch removal, the clipped areas were depilated with Neet Cream Hair Remover (Reckitt & Colman, Wayne, NJ 07474). The depilatory was allowed to remain on the sites for 5-10 minutes and then wiped with a towel moistened with warm tap water. Approximately three (3) hours after depilation the patch sites were scored for the 24 hour grades. Approximately twenty-four (24) hours after the first grading, the second observation was made.

For consistency of evaluations with other denial studies at Stonybrook, the Draize scoring scale was used in order to express the data rather than the Buehler scoring scale.

The Draize scoring scale is shown below:
Erythema and Eschar Formation
0 No erythema
1 Very slight erythema (barely perceptible)
2 Well-defined erythema
3 Moderate to severe erythema
4 Severe erythema (beet redness) to slight eschar formation (injuries in depth)

Induction
Dermal contact with test substance is intended for use as a industrial lubricant base stock at concentrations of 50 to 100%. Since the irritation potential of test substance was unknown, the Primary Irritation Phase was conducted prior to the first induction application. Based on results obtained from the Primary Irritation Phase, a 75.0% w/w concentration in mineral oil was selected as a reasonable induction dose. Mineral oil was selected as the vehicle for test substance based on solubility and historical experimental test data in guinea pigs and humans with petroleum additives (e.g., esters). Mineral oil has been shown to be non- sensitizing in guinea pig and human sensitization studies, therefore, a vehicle control group was not utilized in an effort to conserve the use of animals.

The upper left quadrant of 20 guinea pigs was clipped using electric clippers. On the following day, the patches were applied using a Hill Top Chamber with a 25 mm Webril swatch saturated with 0.3 ml of test substance (75.0% w/w in mineral oil).

The induction guinea pigs were removed from their restrainers and the test substance removed according to the technique described above.

The patches were reapplied to the same site once a week for 3 weeks for a total of three six-hour exposures; test substabce (75.0% w/w in mineral oil) was used for each application. Approximately one hour after patch removal, and again approximately 24 hours later, the induction sites were graded according to the Draize scoring scale.
Four-tenths (0.4) ml of the positive control material (DNCB) was administered at a concentration of 0.05% w/v in 80% ethanol. The technique for clipping, applying and removal of patches used for the positive control induction animals was identical to that for the test substabce animals. Grading of induction patch sites was performed as stated above.

Challenge
Due to inclement weather conditions, the Challenge patch application was performed following a 16-day rest period (Day 30) versus a 14-day rest period (Day 28). The right rear quadrant of the Induction and Challenge Control guinea pigs were clipped two days prior to dosing versus the thy before. The animals did not require any additional clipping. On the following day (first day), a challenge patch containing 0.3 ml of test substance (50.0% w/w in mineral oil) was applied to the animals for approximately 6 hours according to the technique previously described. Approximately twenty-one (21) hours after patch removal, the clipped areas were depilated. The depilatory was allowed to remain on the sites for 5-10 minutes and then wiped with a towel moistened with warm tap water. Approximately three (3) hours later (30 hours from patch application) the patch sites were scored (24 hour reading). Approximately twenty-four (24) hours later a second observation was made (54 hours from patch application).

The mineral oil vehicle control patch was not utilized during challenge according to Section 1.6.2.3.2 of the EEC Commission Directive. This vehicle control site was reserved in the event of a possible rechallenge at which time the vehicle would have been administered.

DNCB (0.4 ml) was administered in the same manner as test substance, but using a 0.05% (w/v) concentration in acetone.
Challenge controls:
Groups of 10 naive control animals were used as negative controls during challenge responses to test substance and DNCB positive control.
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene (DNCB)

Results and discussion

Positive control results:
---Primary Irritation---
Based on historical data generated at Stonybrook Laboratories Inc. (formerly Environmental and Health Sciences Laboratory), the 0.05% w/v concentration of DNCB in acetone has been determined to be the highest nonirritating concentration for challenge.

---Induction (0.05% w/v in 80% ETOH)---
Grading conducted after the first induction patch gave no indication of skin irritation.
However, skin reactions were observed after the second and third induction patches.
Individual animal data are presented in Appendix ifi.

---Challenge (0.05% w/v in acetone)---
When the induced and naive positive control animals were challenged with DNCB, the following results were obtained:
Number of Animals Showing Indicated Ervthema Score
Score: 0 1 2 3 4 Severity
24-Hour Induced 0 1 6 3 0 2.2
Reading Control 9 1 0 0 0 0.1

48-Hour Induced 0 0 8 2 0 2.2
Reading Control 10 0 0 0 0 0.0

The positive control animals were considered to have been sensitized, based on the test criterion of a minimum of a “+2” response for positively-responding animals.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50% test substance
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% test substance. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50% test substance
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% test substance. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% test substance
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: challenged naive control. Dose level: 50% test substance. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% test substance
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: challenged naive control. Dose level: 50% test substance. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.05% DNCB (w/v) in acetone
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.05% DNCB (w/v) in acetone. No with. + reactions: 9.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.05% DNCB (w/v) in acetone
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.05% DNCB (w/v) in acetone. No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.05% DNCB (w/v) in acetone
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: challenged naive control. Dose level: 0.05% DNCB (w/v) in acetone. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.05% DNCB (w/v) in acetone
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: challenged naive control. Dose level: 0.05% DNCB (w/v) in acetone. No with. + reactions: 0.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The frequency of skin sensitizing responses for 1,4-Benzenedicarboxylic Acid Di-C11-14-Isoalkyl Ester, C13-Rich is 0 out of 20 treated guinea pigs. This finding does not warrant the classification of 1,4-Benzenedicarboxylic Acid Di-C11-14-Isoalkyl Ester, C13-Rich as a skin sensitizer under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

1,4-Benzenedicarboxylic Acid Di-C11-14-Isoalkyl Ester, C13-Rich tested in 20 guinea pigs by the Buehler method to assess skin sensitization potential. Clinical observations were made at 24 and 48 hours post challenge. No adverse clinical effects noted. At the end of the study the frequency of positive challenge responses to 50% test substance in mineral oil was 0 out of 20 guinea pigs. This finding does not warrant the classification of 1,4-Benzenedicarboxylic Acid Di-C11-14-Isoalkyl Ester, C13-Rich under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.