Propanoic acid, 2-[(ethoxythioxomethyl)thio]-, methyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

The relative humidity of the animal room was sometimes higher than the upper limit mentioned in the guidelines. The topical induction (pre)treatment was carried out one day later than mentioned in the protocol. These deviations had no impact on the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

In vivo test was made in 2000 before the in vitro test strategies.

Test material

Test material information

Constituent 1

Test material form:

liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

SPF-bred / Crl: (HA) BR

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: circa 3 weeks (ordered)
- Weight at study initiation: no data available
- Housing: in a mobile battery, containing 6 cages; maximal 10 animais per cage.
- Diet (e.g. ad libitum): standard laboratory diet ad libitum. Each batch of this diet is analyzed by the supplier (SOS Special Diets services, Whitham, England) for the nutrients and contaminants and the results are available upon request.
- Water (e.g. ad libitum): Tap water (N.V. Waterleidingbedrijf Midden-Nederland) ad libitum. Results of routine physical, chemical and microbiological examination of drinking water as conducted by the supplier are available upon request.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°c
- Humidity (%): 56.6-99.9% (upper limit higher than 70%, because of wet cleaning of the animal room and/or meteorological circumstances).
- Air changes (per hr): ca 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 hours dark cycle
- IN-LIFE DATES: From: 31 May 2000 To: 30 june 2000

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

maize oil

Concentration / amount:

10% in maize oil (test group)

Day(s)/duration:

Day 1

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

intradermal

Vehicle:

maize oil

Concentration / amount:

100% (control group)

Day(s)/duration:

Day 1

Induction 3

Route:

epicutaneous, occlusive

Vehicle:

maize oil

Concentration / amount:

100% (control group)

Day(s)/duration:

Day 8 / 48 hours

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Induction 4

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100 % (undiluted) (test group)

Day(s)/duration:

Day 8 / 48 hours

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% (undiluted) (test group)

Day(s)/duration:

Day 21 / 24 hours

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% (undiluted) (control group)

Day(s)/duration:

Day 21 / 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Preliminary test: 4 male animals

Main test:
Number of animals in test group: 10
Number of animals in negative control group: 5

Details on study design:

RANGE FINDING TESTS:
The irritation response to intradermal injection of various concentrations of the test substance was examined in 2 guinea pigs. A sufficiently large area of the flanks was clipped free from hair with electric clippers. Amounts of 0.1 ml of the selected concentrations were applied by intradermal injection. Circa 24 hours after injection, the animals were examined for signs of irritation. A concentration causing
slight to moderate irritation but otherwise well-tolerated by the animals, is usually taken for intradermal injection of the test substance in the induction phase of the main study.
The irritation response to topical treatment of various concentrations of the test substance was examined in 2 other guinea pigs. The flanks of each of the animals were clipped free from hair with electric clippers. Patches (Silverpatch, v .d. Bend B.V., Brielle, the Netherlands) were loaded with the test material and placed on the clipped skin of each animal, and covered with a piece of hypoallergenic paper
bandage (Leukopor) that was secured by elastic adhesive bandage (Tensoplast), wound around the torso of the animal. The dressing was left in place for ca 24 hours. Circa twenty-four and 48 hours after removal of the dressing, the animals were examined for signs of skin irritation. A concentration causing slight to moderate skin irritation is usually chosen for topical induction and a non-irritant concentration for topical challenge.

MAIN STUDY
Fifteen male guinea pigs were randomly divided into two groups, viz. one test group of 10 males and one control group of 5 males. The animals were weighed one day before the study was initiated and at the completion of the study.

A. INDUCTION EXPOSURE
Induction was effected in two different ways, firstly by intradermal injections and secondly, one week later, by topical application over the injection sites.
a. lntradermal injections
For this purpose an area of about 24 cm2 of dorsal skin in the scapular region was clipped free from hair with electric clippers. Pairs of intradermal injections (0.1 ml each) were made simultaneously in the clipped area.
The following preparations were injected:
test animals
- two injections with Freund's Complete Adjuvant (FCA) and saline (1: 1),
- two injections with the selected test concentration,
- two injections with the selected test concentration in FCA (1: 1),
control animals
- two injections with FCNsaline (1:1),
- two injections with the diluent,
- two injections with FCNdiluent (1: 1).
Skin readings were made at ca 24 hours after the treatment.

b. Topical application
Seven days after the intradermal injections, the dorsal skin in the scapular region of all test and control animais was closely clipped again, and subsequently treated with a 10% dilution of sodium lauryl sulfate in vaseline (open application) to enhance sensitivity of the skin. On the following day, the induction by topical application was made in this region. The test animais were treated as follows:
A circa 2 x 4 cm patch of Whatman No. 3 MM filter paper was loaded with the selected concentration of the test substance. The loaded patch was placed over the sites of the intradermai injections and was secured.
The dressing was left in place for ca 48 hours. The control animais were similarly treated with empty patches. Skin readings were made directly after removal of the patches.


B. CHALLENGE EXPOSURE
The topical challenge with the test substance was carried out 13 days after the topicai induction as follows:
An area of circa 5 x 5 cm on both flanks of each test and control animal was clipped free from hair. Patches were loaded with the test concentration(s) selected. Subsequently, the loaded patch was placed on the clipped area of the flanks of each test and control animal. The patch was covered with Leukopor bandage, and held in place by Tensoplast for ca 24 hours. Skin readings were made at ca 24 and
48 hours after removal of the patch.

OTHER:
Scoring and evaluation of the results:
The controls should not show any skin reaction. If so, the severity and type of reaction was taken into account upon evaluation of eff ects of the skin reactions exhibited by the test animais. In absence of skin reactions on the test site challenged with the test substance of the controls, the observed skin reactions on the challenged test site of the test animais were considered as signs of sensitization. If skin reactions are observed on the test site of the controls challenged with the test substance or with the vehicle alone or if skin reactions occur on the test site of the test animais challenged with the vehicle alone, the difference in type, severity, and persistence of these reactions were evaluated in order to establish whether or not the skin reactions of the test animais were indeed signs of sensitization. The
positive results were evaluated according to the EC-standards (as published in the Official Journal of the European Communities, L 110 A, Volume 36, 4 May 1993), which states that a substance is considered a sensitizer if 30% or more of the test animais show a positive reaction.

Challenge controls:

yes, as described in details on study design

Positive control substance(s):

yes

Remarks:

formaldehyde (37%)

Results and discussion

Positive control results:

The sensitivity of this test system was checked by means of a positive control study with formaldehyde (37% ). Formaldehyde was selected as a reference because of its known weak sensitization potential in the guinea pig maximization test. The challenge treatment with a 10 and 3% test dilution of formaldehyde (37%) induced positive reactions in, respectively, 100 and 80% of the test animais, whereas no skin reactions were observed in the controls. Therefore, it was concluded that the experimental design and the strain of guinea pigs used for routine sensitization testing are suitable to detect possible sensitization potential of test materials.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

slight erythema only at 24 hours after challenge

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

skin irritation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

skin irritation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

10%

No. with + reactions:

5

Total no. in group:

5

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10%

No. with + reactions:

5

Total no. in group:

5

Any other information on results incl. tables

Preliminary tests :

The degree of irritation observed after intradermal treatment with a 10% test concentration was considered suitable for intradermal treatment during the induction phase. The undiluted test substance was considered acceptable for topical treatment during the induction and challenge phase of the study. Since the test concentration selected was not irritating, it was decided to pretreat the induction test site with a 10% sodium lauryl sulfate solution (SLS) in vaseline.

Main study: All animals remained in good health during the experimental period.

Induction :

The intradermal injections generally caused the following skin reactions:

test animals

- with FCA/saline (1: 1): moderate erythema,

- with the selected test concentration: slight erythema,

- with the selected test concentration in FCA/ diluent ( 1: 1 ): slight or moderate erythema,

control animals

- with FCA/ saline (1: 1 ): moderate erythema,

- with the diluent: no skin reactions,

- with FCA/diluent (1: 1): moderate erythema.

Topical pretreatment with 10% SLS induced erythema in the controls and test animals. After the topical application of the vehicle alone, slight erythema was observed in the controls. After the 48-hour topical application of the selected test concentration, mainly moderate erythema was observed in the test animals.

Challenge:

After the challenge treatment with the undiluted test substance, all 10 test animals showed skin reactions, generally consisting of moderate erythema or severe erythema and swelling, whereas one control showed slight erythema only at 24 hours after challenge.

Table : Dermal reactions elicited by the challenge application of undiluted Rhodixan A-1

 

Skin scores obtained at 24 and 48 hours after removal of the dressing
Animal no.	24 hours	48 hours
Control group
102	0	0
104	0	0
106	1	0
108	0	0
110	0	0
Test group
82	3	3
84	2	2
86	3	3
88	3	3
90	2	2
92	3	3
94	3	3
96	2	2
98	3	3
100	1	2

READING OF SKIN REACTIONS

1. Reading of skin reactions after intradermal injection

0 = no visible changes

1 = slight erythema

2 = moderate erythema

3 = severe erythema; eschar formation (injuries in depth)

2. Reading of skin reactions after topical application (Magnusson)

0 = no visible change

1 = discrete or patchy (slight) erythema

2 = moderate and confluent erythema

3 = intense (severe) erythema and swelling

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the conditions of the test, RHODIXAN A-1 was considered to be a skin sensitiser.

Executive summary:

The sensitisation potential of RHODIXAN A-1 was evaluated in male Dunkin-Hartley guinea pigs according to the maximisation method of Magnusson and Kligman OECD guideline No.406, and in compliance with Good Laboratory Practice.

A preliminary test was conducted with two males to determine concentrations to be used in the main study for intradermal injection induction (concentration causing slight to moderate irritation), induction (concentration causing slight to moderate irritation), and challenge (non-irritant concentration).

 

Ten test and five control guinea pigs were included in the study. Intradermal injections were performed on day the scapular area of the back with 0.1 ml of test substance at 10 % (w/w) in maize oil and Freund’s Complete Adjuvant (FCA). Seven days after intradermal injections, the test article was applied undiluted on the same skin sites under an occlusive dressing for 48 hours, after a topical pre-treatment with 10% sodium lauryl sulphate. The animals were challenged 13 days later with an occlusive cutaneous application of the undiluted test substance for 24 hours on one flank. Control animals received intradermal injections of FCA/isotonic saline, or vehicle followed by cutaneous applications of empty patches during the induction period. Control animals were submitted to the challenge treatment. Cutaneous reactions were evaluated 24 and 48 hours after the challenge application.

 

At challenge with the undiluted test substance, all ten animals showed skin reactions, generally consisting of moderate erythema or severe erythema and swelling. One control showed slight erythema only at 24 hours.

 

Under the conditions of the test, RHODIXAN A-1 is considered a skin sensitiser, and is classified Skin sensitization 1B, H317 according to CLP and GHS criteria.