diethyleneglycyl bis (chloroacetate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-08-22 to 2000-09-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1996

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Test was conducted in 2000

Test material

Test material information

Constituent 1

Reference substance name:

2-{2-[(2-chloroacetyl)oxy]ethoxy}ethyl 2-chloroacetate

Cas Number:

106-78-5

Molecular formula:

C8H12O5Cl2

IUPAC Name:

2-{2-[(2-chloroacetyl)oxy]ethoxy}ethyl 2-chloroacetate

Test material form:

liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Hsd Poc:DH

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH Laboratory Animal Breeders in 33176 Borchen
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF-bred
- Age at study initiation: 3 - 6 weeks
- Weight at study initiation: 295 - 360 grams
- Housing: IV Makrolon® cages, in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. Low-dust wood shavings were used as bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least seven days
- Indication of any skin lesions: none



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3°C (possibly drifting higher at outdoor temperatures above 24°C)
- Humidity (%): 40 - 60 %
- Air changes (per hr): >= 10 times per hour
- Photoperiod (hrs dark / hrs light): Twelve hours; artificial lighting from 6 AM to 6 PM

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

polyethylene glycol

Remarks:

400

Concentration / amount:

Intradermal induction: 5% (= 20 mg test item/animal)
Topical induction: 12% (= 60 mg test item/animal)

Day(s)/duration:

Day 0 Intradermal; one week after intradermal induction topical application for 48 h

Adequacy of induction:

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

Challenge

Route:

other: epicutaneous, using hypoallergenic patch loaded with the test item formulation and held securely in place on the skin with a ORABAND® selfadhesive tape

Vehicle:

polyethylene glycol

Remarks:

400

Concentration / amount:

6% (= 30 mg test item/animal)

Day(s)/duration:

Three weeks after intradermal induction

Adequacy of challenge:

other: non-irritant concentration

No. of animals per dose:

10 per dose and 5 in the control group

Details on study design:

RANGE FINDING TESTS: Based on the results of the preliminary test the concentrations for the main study was determined
Individual Range-finding (Pilot) Test Results:
- Dose-range finding study for intradermal induction: One guinea pig was given intradermal injections twice, in each case, with 0.1 ml of the following test item concentrations: 0%, 1%, 2.5% and 5%. The injection sites were evaluated after 24 and 48 hours
- Dose range-finding study for topical induction:
Three different concentrations and the vehicle were tested in each case on four guinea pigs. The patches moistened with 0.5 ml of the test item formulations or the vehicle were applied to each animal under occlusive conditions for 24 hours. At the end ofthe exposure period, the remaining test item or the vehicle were removed with physiological saline solution and twenty-one hours later the treated areas were shorn. The skin reactions were evaluated 48 hours and 72 hours after the start of the application
- Dose range-finding study for challenge:
One week prior to the challenge, the challenge concentration was determined on 2 guinea pigs in the main study that were treated in the same manner as the control animals during the inductions.
Four patches each loaded with 0.5 ml test item formulation or the vehicle were applied to each animal under occlusive conditions for 24 hours. At the end of the exposure period, the remaining test item was removed with physiological saline solution and twenty-one hours later the treated areas were shorn. The skin reactions were evaluated 48 hours and 72 hours after the start ofthe application.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (first induction: intradermal exposure; second induction: epicutaneous exposure)
- Exposure period: three weeks
- Test groups: 1 (intradermal induction)
1. Injection site: cranial/bilateral complete Freund's adjuvant (DIFCO LAB.) diluted 1:1 with sterile physiological saline solution
2. Injection site: medial/bilateral 5% WARO 318 formulated in polyethylene glycol 400
3. Injection site: caudal/bilateral 5% WARO 318 formulated at equal parts in sterile physiological saline solution and complete Freund's adjuvant.
- Control group: 1
The animals ofthe control group were treated in the same manner as the animals of the test item group; however, the formulations for injection site pairs 2 and 3 did not contain any test item but in pair 2 undiluted vehicle and in pair 3 a 50% formulation of the vehicle in a 1:1 mixture FCN/physiological saline solution.
- Site: Starting behind the nape of the neck, three injections each in a row were made on the left and the right side of the spinal column. The 1st and 2nd injections were made as contiguous as possible and the 2nd and 3rd injections in a distance of about 2 cm.
- Frequency of applications: once
- Duration (epicutaneous): One week after intradermal induction topical induction was performed with an exposure duration of 48 hours
- Concentrations: Intradermal induction: 5% and topical induction 12%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Exposure period: 24 hours
- Test groups: 1
- Control group: 1
- Site: test item and control group: left flank (caudal). A patch loaded only with the vehicle was placed also on the left flank (cranial) as control
- Concentrations of test group: 6%
- Evaluation (hr after challenge): 48 and 72 hours after start of the application

Positive control substance(s):

not specified

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

6%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

see under "Any other infromation on results"

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

6%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

see under "Any other infromation on results"

Remarks on result:

no indication of skin sensitisation

Results 3

Hours after challenge:

48

Group:

negative control

Dose level:

6%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

see under "Any other infromation on results"

Remarks on result:

no indication of skin sensitisation

Results 4

Hours after challenge:

72

Group:

negative control

Dose level:

6%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

see under "Any other infromation on results"

Remarks on result:

no indication of skin sensitisation

Results 5

Group:

positive control

Remarks on result:

not measured/tested

Any other information on results incl. tables

General Examinations:

Appearance and behaviour of the test item group were not different from the control group.

At the end of the study, the mean body weight of the treatment group animals was in the same range than that of the control group animals.

After the intradermal induction (first induction) the animals in the control group showed after 48 hour:

• red wheal with red surrounding

• encrustation

• white wheal with red surrounding

The animals in the test item group showed after 48 hours:

• red wheal with red surrounding

• encrustation

• white wheal with encrustation

After 7 days the following effects were recorded at the injection sites in the control group and in the test item group: wheals and encrustations.

At day 9, directly after removal of the patch of the second induction, the treatment area of the second induction showed skin effects (grade 1) in 3 of 10 animals in the test item group and no skin effects in the control group.

Challenge with a concentration of 6%:

Animal No.	Test item patch		Control patch
Control group
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 item group
6	0	0	0	0
7	0	0	0	0
8	0	0	0	0
9	0	0	0	0
10	1	0	0	0
11	0	0	0	0
12	0	0	0	0
13	1	1	0	0
14	0	0	0	0
15	0	0	0	0

Grading of the Skin Reaction:

The skin reactions were assessed 48 and 72 hours after the start of the application to induce the challenge and for the range-finding studies to establish concentrations for the topical induction and challenge in accordance with the following pattern:

0= No reaction

1 = Slight localized redness

2 = Moderate confluent redness

3 = Severe redness and swelling

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the maximization test and with respect to the evaluation criteria the test item exhibits no skin-sensitization potential.