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Description of key information

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
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to EPA OPPTS 870.2600; OECD 406 guideline; under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time that the study was conducted the M&K maximisation method was considered to be the most widely accepted for worldwide regulatory purposes.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Labs, Inc., Chelmsford, MA
- Age at study initiation: At least 21 days old
- Weight at study initiation: 357.0 - 489.7 grams
- Housing: Housed in groups in suspended stainless-steel cages
- Diet (e.g. ad libitum): TEK Hi-Fiber Guinea Pig diet 2041, Harlan Teklad, Madison, WI ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: Minimum of 5 days under same conditions as the main test

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68 +/- 5
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod: 12 hour light / 12 hour dark

Route:
intradermal and epicutaneous
Vehicle:
cotton seed oil
Concentration / amount:
Intradermal primary irritation: 0.1 ml of 100%, 50%, 10% and 1% diluted in CSO
Topical primary irritation: 0.1 ml of 100%, 75%, 50% and 25% diluted in CSO
Intradermal induction: 50% concentration test material
Topical induction and challenge: 100% concentration test material
Intradermal induction: 50% test material in CSO
Topical induction: 100% test material
Topical challenge: 100% test material

Based on the results of the primary irritation study, the test material was applied at the minimal irritating dose for induction and maximal non-irritating dose for the challenge. The test material caused severe intradermal irritation at 100% and moderate irritation at 50%. The test material when applied topically caused no irritation at 100%. Based on these results:
- The test material is applied at a concentration of 50% at the intradermal induction.
- The test material is applied at a concentration of 100% for the topical and challenge phase.
Route:
epicutaneous, occlusive
Vehicle:
cotton seed oil
Concentration / amount:
Intradermal primary irritation: 0.1 ml of 100%, 50%, 10% and 1% diluted in CSO
Topical primary irritation: 0.1 ml of 100%, 75%, 50% and 25% diluted in CSO
Intradermal induction: 50% concentration test material
Topical induction and challenge: 100% concentration test material
Intradermal induction: 50% test material in CSO
Topical induction: 100% test material
Topical challenge: 100% test material

Based on the results of the primary irritation study, the test material was applied at the minimal irritating dose for induction and maximal non-irritating dose for the challenge. The test material caused severe intradermal irritation at 100% and moderate irritation at 50%. The test material when applied topically caused no irritation at 100%. Based on these results:
- The test material is applied at a concentration of 50% at the intradermal induction.
- The test material is applied at a concentration of 100% for the topical and challenge phase.
No. of animals per dose:
- Experimental: 10 males/ 10 females
- Negative control group: 5 males/ 5 females
- Positive control group: 2 males/ 3 females
- Primary irritation study: Intradermal: 1 male/1 female; Topical: 1 male/1 female
Details on study design:
Primary irritation phase:
Two primary irritation tests were performed with animals pretreated with Freunds Complete Adjuvant (FCA). These animals were injected 24 hours prior to the start of the primary irritation test with 0.1ml of FCA mixed 1:1 with the vehicle. The intradermal primary irritation study was conducted with two animals and were administered concentrations of 100, 50, 10 and 1% diluted in cottonseed oil (CSO). The topical primary irritation study determines whether the test substance is a skin irritant and was applied at concentrations of 100, 75, 50 and 25% for 24 hours.

The test substance would not emulsify in aqueous vehicles but would in CSO. Any dilutions were made with CSO.

Induction - Intradermal (Day 0):
Three pairs of intradermal injections were made on each side of the midline. Injection pairs 1 and 2 were given in close proximity to each other cranially, whereas injection pair 3 was located caudally. The injections sites were within a 2x4 cm area in the following doses:

- Experimental group:
Injection pair 1: 0.1 ml FCA 1:1 with CSO
Injection pair 2: 0.1 ml test substance (50%)
Injection pair 3: 0.1 ml test substance 1:1 with FCA

- Negative control group:
Injection pair 1: 0.1 ml FCA 1:1 CSO
Injection pair 2: 0.1 ml CSO
Injection pair 3: 0.1 ml of 50% formulation of vehicle, 1:1 with FCA

- Positive control group:
Injection pair 1: 0.1 ml FCA 1:1 CSO
Injection pair 2: 0.1 ml 0.1% benzocaine in 95% ethanol
Injection pair 3: 0.1 ml 0.1% benzocaine in 95% ethanol 1:1 with FCA

Induction - Topical (Day 7):
The test material caused irritation and so pretreatment with sodium lauryl sulfate 24 hours before topical induction application was not necessary.

- Experimental group:
Test material (100%) was spread over a 2x4 cm piece of filter paper to saturation. The patch was covered by an impermeable sheet and secured with a non-adhesive bandage, which was wound around the torso of the animal. The dressing was left in place for 48 hours.

- Negative control group:
Animals were exposed to CSO without test material in the same manner as the experimental group.

- Positive control group:
Animals were exposed to 0.1% benzocaine solution in 95% ethanol in the same manner as the experimental group.

Challenge - Topical (Day 21):
Pieces of filter paper (2x2 cm) were secured to the flanks for 24 hours with the same occlusive bandage as for topical induction for the test and negative control animals. The patch applied to the left side was saturated with the test substance in the highest non-irritant concentration (100%) and the patch on the right side was saturated with vehicle only. In the positive control animals, the patch on the left was saturated with 0.1% benzocaine in ethanol instead of test substance. In the negative control animals, patches were saturated in CSO alone.

Skin readings (Day 23 and 24):
After 21 hours of removing the patches, the flank skin was cleaned and shaved. 3 hours later, the first readings of reactions were made and the second readings made the following day.
Challenge controls:
In the positive control animals, a patch is applied to the left flank and is saturated with 0.1% benzocaine in ethanol. In the negative control animals, patches with CSO alone was applied.
Positive control substance(s):
yes
Remarks:
Benzocaine
Positive control results:
The positive control animals showed skin reactions at each observation point (100% sensitised).
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 9.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 7.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1%
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1%. No with. + reactions: 5.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1%
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.1%. No with. + reactions: 5.0. Total no. in groups: 5.0.

Irritation/corrosivity:

In the primary irritation tests, the test material caused severe intradermal irritation at 100% and moderate irritation at 50%. Topically, the test material caused no irritation at 100%. Based on these findings, the test material was applied at a concentration of 50% at intradermal induction and 100% for the topical and challenge phase.

Sensitisation:

9 out of the 20 test animals showed signs of slight erythema at the 24 hour observation point post challenge (45% sensitised). 7 of these 9 animals were still exhibiting slight erythema at the 48 hour observation post challenge. None of the negative control animals showed any reaction to the challenge (0% sensitised).

All animals gained in body weight. No sytemic signs of toxicity were observed in treated or control animals.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Under the conditions of this study, the study author reports that the test material has been determined to be moderately sensitising.
Executive summary:

Prior to the induction phase, two primary irritation tests (intradermal and topical) were performed with animals pretreated with Freund’s Complete Adjuvant (FCA). Two animals were used in the intradermal primary irritation study and an additional two animals were used in the topical primary irritation study. The test material caused severe intradermal irritation at 100% and moderate irritation at 50%. Topically, the test material caused no irritation at 100%. Based on these findings, the test material was applied at a concentration of 50% at intradermal induction and 100% for the topical and challenge phase.

Ten animals of each sex were used in the definitive sensitisation test. For the intradermal induction, three pairs of intradermal injections were made on each side of the midline. Injection 1 consisted of Freund’s Complete adjuvant with vehicle; injection 2 consisted of test material (at a concentration of 50%); injection 3 consisted of test material and vehicle. Intradermal induction in the negative and positive control groups were treated in a similar fashion. Topical induction was performed on day 7, and involved application of filter paper (2x4 cm) saturated in test material (100% concentration) to the flank side and secured by an occlusive bandage. The dressing was left in place for 48 hours. Topical challenge was performed on Day 21, with the application of filter paper saturated in test material (100% concentration) and secured to the flank area by occlusive bandaging for 24 hours. Skin readings were taken 24 and 48 hours post removal of the topical challenge bandage.

9 out of the 20 test animals showed signs of slight erythema at the 24 hour observation point post challenge (45% sensitised). 7 of these 9 animals were still exhibiting slight erythema at the 48 hour observation post challenge. None of the negative control animals showed any reaction to the challenge (0% sensitised). The positive control animals showed skin reactions at each observation point (100% sensitised). All animals gained in body weight. No sytemic signs of toxicity were observed in treated or control animals. Using the Kligman scoring system, the study author concludes that the test material can be considered to be a moderate skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In the key study, the potential of the test material to cause skin sensitisation was assessed in a Guinea Pig Maximisation test conducted in accordance with EPA OPPTS 870.2600 and OECD 406 under GLP conditions.

Prior to the induction phase, two primary irritation tests (intradermal and topical, 2 animals per test) were performed with animals pre-treated with Freund’s Complete Adjuvant (FCA). The test material caused severe intradermal irritation at 100 % and moderate irritation at 50 %. Topically, the test material caused no irritation at 100 %. Based on these findings, the test material was applied at a concentration of 50 % at intradermal induction and 100 % for the topical and challenge phase. Ten animals of each sex were used in the definitive sensitisation test.

For the intradermal induction, three pairs of intradermal injections were made on each side of the midline. Injection 1 consisted of FCA with vehicle; injection 2 consisted of test material (at a concentration of 50 %); injection 3 consisted of test material and vehicle. Intradermal induction in the negative and positive control groups were treated in a similar fashion. Topical induction was performed on day 7, and involved application of filter paper saturated in test material (100 % concentration) to the flank side and secured by an occlusive bandage. The dressing was left in place for 48 hours. Topical challenge was performed on Day 21, with the application of filter paper saturated in test material (100 % concentration) and secured to the flank area by occlusive bandaging for 24 hours. Skin readings were taken 24 and 48 hours post removal of the topical challenge bandage.

Nine out of the 20 test animals showed signs of slight erythema at the 24 hour observation point post challenge (45 % sensitised). Seven of these 9 animals were still exhibiting slight erythema at the 48 hour observation post challenge. None of the negative control animals showed any reaction to the challenge (0 % sensitised). The positive control animals showed skin reactions at each observation point (100 % sensitised). All animals gained in body weight. No systemic signs of toxicity were observed in treated or control animals.

Under the conditions of this study the test material is considered to cause sensitisation.

Migrated from Short description of key information:

Sensitising, Hartley Guinea Pig, EPA OPPTS 870.2600 and OECD 406 (Lister, 2004)

Justification for selection of skin sensitisation endpoint:

Only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to skin sensitisation as Category 1B (H317: May cause an allergic skin reaction).

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance requires classification with respect to skin sensitisation as R43: May cause sensitisation by skin contact.