Registration Dossier

Administrative data

Description of key information

The test article was not sensitizing in a Guinea Pig Maximisation test according to OECD TG 406 performed with pure test article.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT was performed according to guideline and GLP - no further data is necessary
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley Inc., Houston, Texas
- Age at study initiation: no data
- Weight at study initiation: Males 432-476 g; Females 380-495 g (Test Group); Males 420-495 g; Females 405-491 g (Control Group)
- Housing: single housing (cage: suspended, wire bottom, stainless steel)
- Diet: Purina Guinea Pig Chow; ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 30-80
- Air changes (per hr): 10-12
- Photoperiod: 12 hrs dark / 12 hrs light
Route:
intradermal
Vehicle:
cotton seed oil
Concentration / amount:
5%
Route:
epicutaneous, occlusive
Vehicle:
cotton seed oil
Concentration / amount:
patch saturated with moistened test substance
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
cotton seed oil
Concentration / amount:
100%
No. of animals per dose:
Control: 5 animals per sex
Dose groups: 10 animals per sex
Details on study design:
RANGE FINDING TESTS:
A screening test was conducted with 3 males and 3 females prior to the definitive study. Five percent (w/v) test material in cottonseed oil was selected for intradermal injection. For the topical insult, a dose of 500 mg of test material moistened with petrolatum was selected because it was not irritating in the screening test.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous, occlusive)
- Test group:
Intradermal (3 pairs of injections): 1:1 mixture (v/v) FCA/water, test substance (5%) in cottonseed oil, test substance (5%) in a 1:1 mixture FCA/water and cottonseed oil
Epicutaneous: 2 x 4 cm patch of filter paper was saturated with the moistened test material
- Control group:
Intradermal (3 pairs of injections): 1:1 mixture (v/v) FCA/water, Cottonseed oil, 1:1 mixture (v/v) FCA/water and cottonseed oil
Epicutaneous: dry filter paper
- Site: upper back

B. CHALLENGE EXPOSURE
- No. of exposures: 1(epicutaneous, occlusive)
- Day of challenge: day 21
- Exposure period: 24 h
- Test group:
Epicutaneous: 500 mg on a filter paper moistened with petrolatum
- Control group:
Epicutaneous: dry filter paper
- Site: on the flank
- Concentrations: 0 and 500 mg (0 and 100%)
- Evaluation (hrs after challenge): 24 and 48 h

OTHER:
The application site was pretreated with 0.5 mL of a 10% sodium-laurylsulfate (open application) 24 hours prior to the epidermal induction application.
Positive control results:
No positive controls were included in this study. The reference values with DNCB were reported (experimental date May-June 1994).
DNCB (Intradermal induction: Concentration of compound: 0.1%; Vehicle: cottonseed oil; Epidermal induction: Concentration of compound: 1%; Vehicle: vaseline; Epidermal challenge: Concentration of compound: 0.1%; Vehicle: vaseline) induced reactions in 18/18 animals, thus meeting the reliability criteria (>= 15% positive responders).
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
500 mg (100%)
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
500 mg (100%)
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
500 mg (100%)
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
500 mg (100%)
No. with + reactions:
0
Total no. in group:
20

Erythema and edema reactions (score 1-2) have been observed after intradermal induction (day 6) at the sites treated with Freund´s Complete Adjuvant. No reactions have been observed at the sites treated with test substance alone, neither during induction nor challenge period.

Interpretation of results:
GHS criteria not met
Conclusions:
Since none of the test animals (Group I) or control animals (Group II) exhibited skin reactions after the challenge treatment, the test material was rated non-sensitizing according to the methods of Magnusson and Kligman.
Executive summary:

A maximization test for topically applied test materials was conducted on 30 short-haired male and female albino guinea pigs to determine if the test mnaterial produced a sensitizing reaction. This study was designed and performed in accordance with OECD Guideline 406, EPA FIFRA Guideline 81-6, EPA TSCA (40 CFR 798.4100), and EPA Good Laboratory Practice Standards (40 CFR 792). Methods and grading of sensitization were based on Magnusson and Kligman (J. Invest. Dermat. 52: 268-276 (1969)). Twenty guinea pigs (Group I) each received three pairs of intradermal injections (adjuvant, a 5 % w/v solution of the test material, and a mixture of adjuvant and 5% w/v solution of the test material) followed one week later by a single topical application of 500 mg of test material moistened with petrolatum. The 5% dose was determined from pretest screening and is the maximum level suggested (Magnusson and Kligman, 1969), and the 500 mg of test material moistened with petrolatum was chosen for the topical applications because the screening did not indicate excessive irritation. Ten additional animals served as a control group. Control animals (Group II) were treated at the same time periods and locations as the test group but with the vehicle used in place of the test material for the intradermal injections. The control animals received a topical application of dry filter paper only. Two weeks after the topical application, all animals were challenged with a topical application of 500 mg of test material moistened with petrolatum at a virgin test site. The percentage of test animals exhibiting erythema and/or edema after the challenge treatment was used to assign the test material a sensitization potency rating. Since none of the test animals (Group I) or control animals (Group II) exhibited skin reactions after the challenge treatment, the test material was rated non-sensitizing according to the methods of Magnusson and Kligman.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Two studies are available investigating the skin sensitizing properties of the test substance.

In the key study (Stillmeadows, 1994), a maximization test was conducted on 30 short-haired male and female albino guinea pigs in accordance with OECD Guideline 406, and EPA Good Laboratory Practice Standards. Methods and grading of sensitization were based on Magnusson and Kligman (1969). During induction, twenty guinea pigs each received three pairs of intradermal injections (adjuvant, a 5 % w/v solution of the test material, and a mixture of adjuvant and 5% w/v solution of the test material) followed one week later by a single topical application of 500 mg of test material moistened with petrolatum. The 5% dose was determined from pretest screening and the 500 mg of test material moistened with petrolatum was chosen for the topical applications because the screening did not indicate excessive irritation. Ten additional animals served as a control group and were treated at the same time periods and locations as the test group but with the vehicle instead of the test material for the intradermal injections. The control animals received a topical application of dry filter paper only. Two weeks after the topical application, all animals were challenged with a topical application of 500 mg of test material moistened with petrolatum at a virgin test site. The percentage of test animals exhibiting erythema and/or edema after the challenge treatment was used to assign the test material a sensitization potency rating. Since none of the test animals or control animals exhibited skin reactions after the challenge treatment, the test material was rated non-sensitizing according to the methods of Magnusson and Kligman.

In a second Guinea Pig Maximization test according to OECD TG 406 and in compliance with GLP (Ciba-Geigy, 1988), ten animals per sex in the control group and in the test group were used. The induction was a two-stage operation. First, intradermal injections (into the neck region); second, closed patch exposure over the injection sites one week later. Three pairs of intradermal injections (0.1 ml per injection) were made simultaneously into the shaved neck of the guinea pigs as follows: adjuvant and saline (1:1), 1% test compound in sesame oil, 1% test compound in the adjuvant saline mixture. One week later the test material (30%) was incorporated in vaseline and applied on a filterpaper patch to the neck of the animals (patch 2 x 4 cm; occluded administration for 48 hours). Two weeks after the epidermal induction application the animals were tested on the flank with test substance in vaseline (10%) and the vehicle alone (patch 2 x 2 cm; occluded administration for 24 hours). A control group was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test compound (at least 10 animals) to control the maximal subirritant concentration of the test compound in adjuvant treated animals. No animals with reactions were observed in the control group. In the test group, 9/20 animals showed skin reactions at 24 and 48 h after challenge procedure (Erythema and edema; score 1 -2). Thus, the test material did show sensitizing properties in the guinea pig maximization test.

The result of the second study contradicts the findings observed in the key study. However, the older study was performed with test material containing up to 1% of N-phenyl-naphthylamine, which is known to be a strong sensitizer. It is believed that the sensitizing properties of the test substance reported in the study from 1988 was due to the high content of N-phenyl-napththylamine. In recent years the manufacture of the test material has improved resulting in batches with higher purity and less content of N-phenyl-napththylamine. As a result, the maximization test conducted at Stillmeadows in 1994 was conducted with a batch containing < 0.2 % (Stillmeadow, 1994) of N-phenyl-napththylamine. This batch did not cause any skin reactions in test animals. In a more recent analytic examination (Ciba, 2000) the content of N-phenyl-napththylamine was reduced even further to an amount of below 0.1%. In another substance analysis from 2008 (Ciba, 2008), the content of N-phenyl-napththylamine was measured at 0.03 %. This data shows that over the years the manufacture of the product has improved extensively and purity has increased to over 99% with very little contamination with N-phenyl-napththylamine. Therefore, and based on the key study performed with test substance of higher purity, the test article is considered to be not sensitizing and does not require classification.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for sensitization is not warranted under Regulation (EC) No.1272/2008.