Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

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:
25 November 2003 to 20 December 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Remarks:
US EPA GLP, 40 CFR, Part 792 and OECD GLP
Type of study:
Freund's complete adjuvant test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Freund's complete adjuvant test has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.

Test material

Constituent 1

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Male and female Hartley guinea pigs weighed 302.2 to 496.4 g and were at least 21 days old. They were group-housed upon arrival in stainless steel suspended cages. The animals were acclimated for at least 5 days prior to dosing. Water and feed were provided ad libitum. The temperature and humidity were maintained at 68 ± 5 °F and 30 - 70 %, respectively. Room lights were on a 12-hour light/dark cycle.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
50% test substance for intradermal induction
100% for topical induction (occlusive)
Day(s)/duration:
Intradermal induction on Day 0, topical induction on Day 7 (48 hour exposure)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
75% for topical challenge
Day(s)/duration:
Challenge on Day 21 (24 hour exposure)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10/sex Test group (50% test substance intradermal; 100% test substance topical induction and 75% test substance topical challenge)
5 males/5 females Negative control group
2 males/3 females Positive control group
Primary irritation: 1/sex each for intradermal and topical
Details on study design:
The application sites were prepared by clipping the skin of the test site free of hair. On day 0 and day 7, an approximately 5 x7 cm area over the shoulder region was prepared. On day 21, an approximately 4 x 4 cm square of the flank was prepared.

Before the start of the Induction Phase, two Preliminary Irritancy tests were conducted, in which it was determined that 50% test substance in water was the highest intradermal dose that did not illicit strong local or systemic reactions. The test substance was applied at the minimal topical irritating concentration, 100%, for the topical induction. For the topical challenge, the test substance was applied at the highest non-irritating concentration, 75% test substance in water.


On Day 0, for the intradermal induction, three pairs of intradermal injections were made so that on either side of the midline in the shoulder region there were three injection sites. The six injection sites were just within the boundaries of 2 x 4 cm. The dosing solutions were as follows:

Experimental Group
Injection Pair 1: 0.1 mL FCA 1:1 with Water for Injection
Injection Pair 2: 0.1 mL test substance
Injection Pair 3: 0.1 mL test substance 1:1 with FCA

Negative Control Group
Injection Pair 1: 0.1 mL FCA 1:1 with Cottonseed Oil (CSO)
Injection Pair 2: 0.1 mL CSO
Injection Pair 3: 0.1 mL 50% formulation of vehicle 1:1 with FCA

Positive Control Group
Injection Pair 1: 0.1 mL FCA 1:1 with CSO
Injection Pair 2: 0.1 mL 0.1% Benzocaine in 95% EtOH
Injection Pair 3: 0.1 mL 0.1% Benzocaine in 95% EtOH 1:1 with FCA

The benzocaine was dissolved in 95% ethanol and suspended in FCA to a final concentration of 0.1%.

On Day 7, for the topical induction, the experimental group was dosed with 100% the test substance. As the test substance was an irritant, the area was not pretreated with 0.5 mL of 10% sodium lauryl sulfate 24 hours prior to topical induction application. The test substance was spread over a 2 x 4 cm piece of filter paper in a thick even layer. The patch was placed on the dorsal surface of the animals, 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. The negative control group was treated in a similar fashion with CSO instead of the test substance. The positive control group was exposed to 0.1% Benzocaine in 95% EtOH instead of the test substance.

On Day 21, for the topical challenge, pieces of filter paper measuring 2 x 2 cm were secured to the flanks for 24 hours, utilizing the same wrapping technique as previously described for the topical induction. For both the test and negative control groups, one patch was placed on the left side with the test substance at 75% and the second patch was placed on the right side with the vehicle alone. For the positive control animals, the patch on the left side was saturated with 0.1% Benzocaine in ethanol instead of the test substance.

Approximately 21 hours after patch removal, the flank skin was cleaned and clipped. Approximately 3 hours later, the first reading of the reactions was performed (Day 23) and the second reading was made on Day 24. For evaluation of skin reactions, the following four-point scale was used:

0 = No reactions
1 = Discrete or patchy erythema
2 = Moderate and confluent erythema
3 = Intense erythema and swelling

Daily observations were made for clinical signs of toxicity. Animals were weighed at the beginning and end of the observation period (Days 0 and 24).
Challenge controls:
Negative control: Cottonseed Oil
Positive contro: Benzocaine
Positive control substance(s):
yes
Remarks:
Benzocaine (CAS # 94-09-7); white solid

Results and discussion

Positive control results:
The positive control animals exhibited skin reactions at each observation point (100% sensitized).

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
No systemic signs of toxicity were observed
Remarks on result:
other: 75% sensitisation rate
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
13
Total no. in group:
20
Clinical observations:
No systemic signs of toxicity were observed
Remarks on result:
other: 65% still appearing sensitised
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
CSO
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No systemic signs of toxicity were observed
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
CSO
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No systemic signs of toxicity were observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1% Benzocaine in 95% EtOH
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
No systemic signs of toxicity were observed
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1% Benzocaine in 95% EtOH
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
No systemic signs of toxicity were observed
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

All animals gained weight over the course of the study.  No systemic signs of toxicity were observed in any animal over the course of the study.  None of the negative control animals exhibited any reaction to the challenge.  The positive control animals exhibited skin reactions at each observation point (100% sensitized).

In the main study, 15/20 animals (75% sensitization rate) exhibited discrete to moderate erythema at the 24 hour observation following the challenge application.  At 48 hours following the challenge application, 13/20 were still exhibiting signs of erythema.

Skin Examination Results for the Test Group Challenged at 75% test substance

Animal Number

Sex

Hours After Challenge

24 Hours

48 Hours

1

Male

1

1

2

0

0

3

2

1

4

0

0

5

0

0

6

2

1

7

1

1

8

1

1

9

1

1

10

0

0

11

Female

1

1

12

2

1

13

1

1

14

2

0

15

1

1

16

0

0

17

2

1

18

1

1

19

2

2

20

1

0

Applicant's summary and conclusion

Interpretation of results:
other: EU Criteria: Category 1: May cause an allergic skin reaction.
Conclusions:
Under the conditions of this study, the test material is considered to be a strong skin sensitizer as 75 % of the animals treated exhibited discrete to moderate erythema at the Challenge exposure following an Induction Phase.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 406 and EPA OPPTS 870.2600, under GLP conditions. 

Based on the results of the Primary Irritation study, the test material was applied at the minimal irritating dose for induction and the maximal non-irritating dose for challenge.

Under the conditions of the study, the test material elicited discrete to moderate erythema to the Challenge in the skin sensitisation Kligman Maximisation Test.  Fifteen out of twenty of the test animals (75 % sensitisation) exhibited discrete to moderate erythema at the Challenge exposure following an Induction Phase and therefore the test material is considered to be a strong sensitiser.

All animals gained weight over the course of the study.  No systemic signs of toxicity were observed in any animal over the course of the study.  None of the negative control animals exhibited any reaction to the challenge.  The positive control animals exhibited skin reactions at each observation point (100% sensitized).