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Description of key information

WoE; Denton (1994), performed to GLP and EU method B.6, Klimisch 1, non-sensitising.

WoE; Hutchinson (2002c) performed to GLP, OECD 406, EU Method B.6 and EPA OPPTS 870.2600, Klimisch 1, non-sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16th Februrary to 22nd March 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study is over 12 years old and precludes LLNA method
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D.Hall, Newchurch, Staffordshire, England.
- Age at study initiation: 6 - 7 weeks old.
- Weight at study initiation: 291 to 341g.
- Housing: In groups of 5 in suspended metal cages with wore mesh floors.
- Diet (e.g. ad libitum): Vitamin C enriched guinea-pig diet FDI, ad libitum. Hay provided weekly.
- Water (e.g. ad libitum): drinking water, ad libitum.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21ºC.
- Humidity (%): 30-70 %.
- Air changes (per hr): 15 charges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of artificial light (0700 - 1900) in each 24 hour period.
Route:
intradermal and epicutaneous
Vehicle:
other: Alembicol D a product of coconut oil.
Concentration / amount:
Induction intradermal injection: 0.25 w/v in Alembicol D
Induction topical application: 60% w/v in Alembicol D
Challenge application: 60 and 30% w/v Alembicol D
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D a product of coconut oil.
Concentration / amount:
Induction intradermal injection: 0.25 w/v in Alembicol D
Induction topical application: 60% w/v in Alembicol D
Challenge application: 60 and 30% w/v Alembicol D
No. of animals per dose:
10 test and 5 control animals.
Details on study design:
RANGE FINDING TESTS: A preliminary study was performed to identify a suitable concentration for the main test.

MAIN STUDY
A. INDUCTION EXPOSURE
- The concentration used was the maximum practical concentration.
- Site: An area of 40 x 60 mm on the dorsal was prepared by clipping the hair.

Intradermal Injection:
- No. of exposures: 3 intradermal injections per site.
- Area of site: 2 x 4 cm.
- Injection 1 = Freud's complete adjuvant, diluted with equal volume of water for irrigation.
- Injection 2 = 0.25% w/v of the test material in Alembicol D.
- Injection 3 = 0.25 w/v of the test material in a 50:50 mixture of Freud's complete adjuvant and Alembicol D.

Topical application:
- 6 days after the intradermal injections the area was prepared by clipping the hair and rubbing with 0.2 ml of 10% w/w sodium lauryl sulphate in petrolatum. 24 hours later a patch saturated in 0.4 ml of the test material, 60% w/v in Alembicol D, was attached to the test site. The patch was fixed with an impermeable adhesive tape.
- Exposure period: 48 hours.
- Area of site: 20 x 40 mm.

- Control group:
Animals were treated the same as in the induction phase, except for the test material was omitted.

B. CHALLENGE EXPOSURE
- 2 weeks after the topical induction both the control and the test group were exposed to the test material. The site was prepared by clipping the hair and a patch saturated in 0.2 ml of the test material was fixed at two sites, the anterior flank and posterior.
- Exposure period: 24 hours.
- Site area: 20 x 20 mm
- Concentration: 60% w/v at the anterior site and 30% w/v to the posterior site.
- Evaluation (hr after challenge): 24, 48 and 72 hours after the removal of the patches.

OBSERVATION:
- Clinical signs: all animals were observed daily.
- Bodyweight: Recorded on Day 1 and on the last day of the observation period.
- Dermal response: Scored according to the Draize scale (1997) which can be seen in table 1 in the field "any other information on materials and methods incl. tables".

INTERPRETATION OF RESULTS
- Positive reaction criteria: If the reaction at challenge was more marked and/or persistent than the maximum reaction seen in the control group, the result would be considered positive.
- Inconclusive reaction criteria: If the reaction was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result is considered inconclusive.
- Negative reaction criteria: If the dermal reaction resulting from the challenge application was the same as or less marked and/or persistent than the maximum reaction seen in the control animals, the result is considered negative.
Challenge controls:
Same as test challenge.
Positive control substance(s):
yes
Remarks:
Formalin
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
60% w/v anterior site
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
30% w/v posterior site
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
60% w/v anterior site
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
30% w/v posterior site
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation

Clinical Signs:

No signs of ill health or toxicity were recorded.

Bodyweight:

All animals showed the expected increase in bodyweight.

Intradermal Injections:

Necrosis was recorded at sites receiving Freund's Complete Adjuvant in the test and control animals.

Slight irritation was seen in test animals at sites receiving 0.25% w/v in Alembicol D and was also observed in control animals receiving Alembicol D.

Topical Application:

Very slight erythema was observed in test animals following topical application with 60% w/v in Alembicol D.

Very slight erythema was also seen in the control animals.

Challenge:

There was no dermal reaction seen in any of the test or control animals.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the test the test material was determined to be not sensitising.
Executive summary:

In a GLP compliant study which was performed according to the standardised guideline EU Method B.6, the potential for the test material to cause skin sensitisation was determined in a Guinea-pig maximisation test. Ten Guinea-pigs were exposed to the test material, none of which displayed a dermal reaction. No other signs of toxicity were observed in any animal.

Under the conditions of the test the test material is considered to be non-sensitising and therefore according to Regulation (EC) No. 1272/2008 no classification is required.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01 April 2002 - 13 May 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study is over 12 years old and precludes LLNA method
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Darley Oaks, Newchurch, Burton-on-Trent, Staffordshire. Animals arrived on 25 March 2002.
- Age at study initiation: Less than 6 months old (young adult).
- Weight at study initiation: 449 - 507 g in the test group and 486 - 554 g in the control group.
- Housing: Housed in groups of 5, in cages measuring approximately 48 x 61 x 25 cm.
- Diet: Guinea Pig Diet FD1, supplied by Special Diets Services Ltd. The feed was supplied ad libitum, supplemented with hay twice weekly.
- Water: Supplied ad libitum from the main domestic supply.
- Analysis for significant contaminants was performed on both the diet and water supplied to the test animals. Both were considered not to contain any additional substances in sufficient concentrations to have any influence on the outcome of the study.
- Acclimation period: For at least 5 days prior to study initiation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Mean minimum and maximum temperatures were 21 ºC and 20 ºC, respectively.
- Humidity (%): Mean relative humidity was 54%, ranging from 31 to 66%.
- Air changes (per hr): Minimum of 15 air charges per hour.
- Photoperiod (hrs dark / hrs light): A 12 hour light/dark cycle was maintained, where the light hours were between 0700 and 1900 hrs.
Route:
intradermal and epicutaneous
Vehicle:
maize oil
Concentration / amount:
Intradermal injection at 1% solution of the test material in maize oil. A 50% solution of the test material was used for the epicutaneous application.
Route:
epicutaneous, occlusive
Vehicle:
maize oil
Concentration / amount:
Intradermal injection at 1% solution of the test material in maize oil. A 50% solution of the test material was used for the epicutaneous application.
No. of animals per dose:
Ten animals were used in the test group, and 5 for the control.
Details on study design:
RANGE FINDING TESTS:
-Dose range finding test for induction:
Intradermal injections: The test sites were prepared prior to exposure, by clipping. Intradermal injections were tested at four different concentrations 5, 3.5, 2 and 1%. Two animals were used, each receiving two concentrations applied at opposing sites. Animals were injected with 0.1 mL of the appropriate solution in either the upper left or right scapula.
Topical application: Topical applications were applied at 50, 25, 10 or 5% solutions. Two animals were exposed using both flanks as test sites. The hair was clipped free prior to exposure and the test material was applied under an occlusive dressing. Patches were removed after 48 hours and wiped clear with distilled water.
- Rationale for concentration selection: The concentrations were selected where the highest caused mild to moderate skin irritation but were not high enough to cause necrosis or severe reactions.
-Dose range finding test for challenge:
Intradermal injections: The test sites were prepared prior to treatment, by clipping. The following day each animal received 6 intradermal injections according to the same schedule set out for the control in the main test, see Table 1.
Topical application: Six days after animals received the injections the test site was again prepared by clipping and 0.5 mL of a 10% solution of sodium lauryl sulphate was applied. The following day each animal was treated with one topical application of the vehicle, 0.5 mL under an occlusive wrap. The patch was removed after 48 hours and the site wiped with maize oil.
Challenge application: Eleven days after induction the test site was prepared for exposure by clipping. The following day animals were exposed to a topical application of the test material in 50, 25, 10 and 5% solutions. 0.5 mL of each solution was applied under a patch and occluded by wrapping the torso. The patches were removed after 24 hours and the site wiped with maize oil.
- Rationale for concentration selection: The highest dose which did not cause skin irritation.

MAIN STUDY
A. INDUCTION EXPOSURE
- Intradermal induction: Six 0.1 mL intradermal injections were made at the test site, the schedule for which can be seen in Table 1.
- Topical application: The site was prepared 6 days after intradermal induction by clipping the hair and treating the area with 0.5 mL of 10% solution of sodium lauryl sulphate (prepared in distilled water). Topical applications were made on the following day with 0.5 mL of the test material applied under an occlusive dressing for 48 hours. On patch removal the site was wiped clean with maize oil.
- Control group: Control groups were treated in the same manner, except the test material was omitted and replaced with maize oil.
- Site: Scapular region

B. CHALLENGE EXPOSURE
- No. of exposures: one exposure was made 14 days after the topical induction application.
- Exposure period: The test material was removed after 24 hours, application sites were wiped with maize oil after patch removal.
- Test groups: The test group received two topical applications, either 0.5 mL of maize oil on the upper left flank or 0.5 mL of the test material on the upper right flank.
- Site: The site was prepared 13 days after topical application by clipping the hair on both flanks.
- Evaluation: Sites were examined 24 and 48 hours after challenge.
- Scoring:
No visible change = 0
Discrete or patchy erythema = 1
Moderate or confluent erythema = 2
Intense erythema and swelling = 3
- Other observations:
> Viability: Checked twice daily.
> Clinical observations: All animals were examined for reactions to treatment. The onset, intensity and duration of any signs were recorded.
> Body weights: Measured prior and post exposure.
> Necropsy: All animals were sacrificed as soon as possible after the final observations were made.
Challenge controls:
- Concentrations: 0.5 mL of the test material was applied in a 50% solution with maize oil on the upper right flank. 0.5 mL of maize oil was applied to the upper left flank in the same manner as the test group.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole (MBT), experimentally completed in December 2001.
Positive control results:
Following challenge application with MBT at a dose level of 25% w/v in maize oil, 100% of the test animals and none (0%) of the control group reacted positively.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None, other than skin reactions.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None, other than skin reactions..
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None, other than skin reactions.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None, other than skin reactions..

Dose Range Finding Results

-Intradermal induction injections: Moderate to discrete reactions were noted at 1%, higher concentrations proved difficult to administer. Based on these results a 1% concentration was selected for the definitive test.

-Topical induction: No reaction was observed at concentrations up to 50%, thus this was selected for topical induction in the definitive test.

-Challenge: No reaction was noted in either animal up to 50%, thus this was selected for challenge application in the definitive test.

Definitive Test

Body weights: Weight gain is considered to be satisfactory.

Table 2. Sensitisation Results

Group

Animal

% of Test Material/Time after patch removal (hr)

% of Test Material/ Response

50%

0%

50

0

24 hrs

48 hrs

24 hrs

48 hrs

Control

1

0

0

0

0

-

-

2

0

0

0

0

-

-

3

0

0

0

0

-

-

4

0

0

0

0

-

-

5

0

0

0

0

-

-

Test Group 1

1

0

0

0

0

-

-

2

0

0

0

0

-

-

3

0

0

0

0

-

-

4

0

0

0

0

-

-

5

0

0

0

0

-

-

Test Group 2

1

0

0

0

0

-

-

2

0

0

0

0

-

-

3

0

0

0

0

-

-

4

0

0

0

0

-

-

5

0

0

0

0

-

-

Table 3. Body Weight

Group

Animal

Body weight (g)

Start of Study

End of Study

Gain

Control

1

497

623

126

2

516

666

150

3

554

691

137

4

501

630

129

5

486

580

94

Test Group 1

1

482

589

107

2

491

613

122

3

449

534

85

4

484

593

109

5

485

545

60

Test Group 2

1

471

602

131

2

507

594

87

3

483

634

151

4

479

556

77

5

476

592

116

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, no animal displayed delayed hypersensitivity to the test material, and thus the test material was determined to be non-sensitising.
Executive summary:

The delayed contact hypersensitivity of the test material was assessed in a maximisation study performed to GLP and the standardised guidelines OECD 406, EU Method B.6 and EPA OPPTS 870.2600.

The test consisted of two procedures induction and challenge. The induction step consisted of exposure via two routes, intradermal injection at 1% and topical application at 50%. The animals were also exposed to an adjuvant material via intradermal injection. This was followed by a challenge to the test material via topical application at 50%. Ten guinea pigs were exposed in the test group and 5 were used as a control. Solutions were prepared in maize oil, where the control group were exposed to the vehicle.

Under the conditions of the test, no animal displayed delayed hypersensitivity to the test material, and it was therefore determined to be non-sensitising.

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

Justification for selection of skin sensitisation endpoint:

This endpoint has been addressed on a weight of evidence basis and thus one study cannot be selected over the other. Denton (1994) and Hutchinson (2002c) were performed according to GLP and in line with standardised guidelines. Both studies were performed to a good standard with a sufficient level of detail to assess the quality of the data presented. They were assigned a reliability score of 1, in accordance with Klimisch (1997) and considered suitable to fulfil the data requirement. The test material was determined to be non-sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification as a skin sensitizer.