Uridine, 2'-deoxy-5-ethyl-, 3',5'-bis(4-chlorobenzoate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 31st, 2000 - February 15st, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

Animals were housed, in groups of up to 10 animals and not 5. Animals were not placed into treatment groups by random numbers.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

In vivo skin sensitisation studies that were carried out or initiated before 11 October 2016, and that meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4).

Test material

Test material information

Constituent 1

Reference substance name:

Uridine, 2'-deoxy-5-ethyl-, 3',5'-bis(4-chlorobenzoate)

EC Number:

607-566-3

Cas Number:

25137-84-2

Molecular formula:

C25H22Cl2N2O7

IUPAC Name:

Uridine, 2'-deoxy-5-ethyl-, 3',5'-bis(4-chlorobenzoate)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2'DEOXY-5-ETHYL-URIDINE, 3', 5'-BIS (4- CHLOROBENZOATE)
- Description : solid (whitish crystalline powder)
- Lot number : β-CEDU 2/98-G
- Expiry date: June 2001
- Storage conditions : at room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Nossan S.r.l., Correzzana (MI)
- Females nulliparous and non-pregnant: [yes/no/not specified] Yes
- Age at study initiation: to 5 weeks of age
- Weight at study initiation: 300 to 350 grams
- Housing: Steel cages, measuring 48 x 63 x 41 cm, with a grid floor
- Diet: laboratory diet (Altromin MSK, Altromin, D-32770 Lage, Postfach 1120 - Germany) ad libitum
- Water: ad libitum
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light):12 hours light/12 hours dark.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

intradermal induction: 2% test substance
epicutaneous induction: 50% test substance
challenge: 20% test substance

Challenge

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

intradermal induction: 2% test substance
epicutaneous induction: 50% test substance
challenge: 20% test substance

No. of animals per dose:

20 animals in the tratment group and 10 animals in the control group were used

Details on study design:

RANGE FINDING TESTS
Intradermal injection: two guinea pigs were treated with 0.1 mL of the test substance concentrations 20%, 10%, 5%, 2%, 1% and 0.5% in corn oil. The physical properties of the substance at the 2 higher concentrations (20% and 10%) prevented intradermal injection. The six treated sites of each animals were examined 6 days later and observed irritation was recorded using the Draize scoring scale.
The preliminary tolerance test indicated that the test item at 2% concentration should be reasonably tolerated and easily injected.
Topical application: 5 guinea pigs were injected intradermally at scapulae area with 0,1 ml of the test substance concentrations 50%, 20%, 10%, 5% and 1% in corn oil under occlusive conditions for 24 hours. Skin reactions were evaluated 24 and 48 hours after removal of the dressings.
The topical application test indicated that the test item at 50% concentration should be tolerated by the test system. A lower concentration of 20% was judged to be non-irritant and was selected for use at challenge

MAIN STUDY
The procedure may be considered in two part: Induction and Challenge

Induction
Induction intradermal injection
A 20 x 40 mm area of skin on the scapular region of the guinea-pig was clipped free of hair using electric clippers. Three pairs of intradermal injections were made within the clipped area. All injections were made at the edge of the prepared site and the anterior and median injections were positioned dose together and distant from the posterior injections. A volume of 0.1 ml was injected at each point.
Animals of the test group were treated as follows:
Injection site Treatment
Anterior Emulsified Freund's complete adjuvant
Median 2% test item in corn oil
Posterior 2% test item in emulsified Freund's complete adjuvant

Animals of the control group were treated in the same manner except that the test item was replaced by the vehicle alone. The treatment plan was:
Injection site Treatment
Anterior Emulsified Freund's complete adjuvant
Median Vehicle (corn oil)
Posterior Vehicle mixed with emulsified Freund's complete adjuvant
Sites were observed approximately 24 hours after injections.

Induction topical application
Six days after injection (Day 7 of the study) the area surrounding the injection sites on each animal was clipped free of hair and the injection site was pre-treated with 0.5 ml aliquot of sodium laurylsulphate at a concentration of 10% in petrolatum.
The next day (Day 8 of the study) animals of the test group were treated with the test item at 50% concentration. A gauze patch was covered with 0.4 ml of the substance and this placed over the injection sites, with the substance in contact with the skin, and covered with a strip of aluminium foil to serve as an occlusive barrier. The animal was then wrapped with a length of elastic adhesive bandage to maintain the gauze patch in contact with the skin. All animals of the test group were treated in this manner. Animals of the control group were similarly treated with the vehicle alone (corn oil). After a contact period of 48 hours the dressings were removed and the treated sites gently cleaned by washing with warm water.
Reaction to treatment was assessed approximately 24 hours after removal of the dressings

Challenge
Three weeks after the intradermal induction, animals were prepared for challenge by clipping the flanks free of hair to expose areas approximately 50 mm x 50 mm on each flank. Patches of gauze measuring 20 mm x 20 mm were coated with 0.2 ml aliquots of the test item at 20% concentration. These were placed on the right flank of each animal, of both test and control groups, in the centre of the prepared skin site. The left flank of each animal was similarly treated with patches coated with 0.2 ml of the vehicle alone (corn oil). The treated sites were covered with a strip of aluminium foil to act as an occlusive barrier and each animal then wrapped with a length of elastic adhesive bandage to keep the test item and vehicle in contact with the skin. After a contact period of 24 hours the dressings and patches were removed.
Approximately 21 hours after removal of the dressings and patches, the treated sites were closely clipped to remove any hair that may have grown. Approximately 3 hours later, 24 hours after removal of the dressings, the treated sites were examined for any signs of reaction to treatment. The examination was repeated 24 hours later.
EVALUTATION CRITERIA: The test would be considered positive if 30% or more of animals in thE test group exhibited erythema or dermal swelling following challenge with a non-irritant concentration of the testitem. The non-irritant nature of the test item at the concentration used at challenge would be demonstrated by the lack of dermal responses in the control group.

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

20

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

20

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

20 %

No. with + reactions:

0

Total no. in group:

10

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

20 %

No. with + reactions:

0

Total no. in group:

10

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained in this study indicate that the test item, 2’deoxy-5-ethyl-uridine,3’,5’-bis(4-chlorobenzoate), does not elicit a sensitisation response in the guinea pig, there being no evidence of response at challenge following a period of induction exposure to the substance.

Executive summary:

The potential of the 2’deoxy-5-ethyl-uridine,3’,5’-bis(4-chlorobenzoate) to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model using the maximisation test of Magnusson and Kligman.

The concentrations of the test item used in the main study were determined by the results of preliminary screening tests. The main sensitisation test was undertaken using a test group of 20 animals and a control group of 10 animals. In an attempt to induce sensitisation, test animals were intradermally injected with an emulsion of Freund's complete adjuvant and the test item at 2% concentration in both the selected vehicle (corn oil) and an emulsion of Freund's complete adjuvant. One week later this was boosted by topical application of the test item at 50% concentration over the injection sites. Control group animals were treated in the same manner but thè selected vehicle (corn oil) was used in place of the test item. Two weeks after the second induction stage, all animals were challenged by topical application of both the vehicle (corn oil) and the test item at 20% concentration.

At the challenge with the test item at 20% concentration no response to the test item was apparent in any animal of either test or control groups. No reaction to the vehicle alone was observed in any animal of either test or control group.