7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 1991 to 16 October 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: method described by Bertil Magnusson, M.D., and Albert M. Kligman, M.D. Ph.D. in "The Identification of Contact Allergens by Animal Assay. The Guinea Pig Maximization Test, " Journal of Investigative Dermatology, 57: 268-276 and in Allergic Contact Dermat

Principles of method if other than guideline:

In accordance with the guideline methods described as OECD 406 or EC B.6 Method

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study predates the validation and adoption of the LLNA as OECD TG 429.

Test material

Test material information

1

Reference substance name:

Cycloaliphatic Epoxy Resin ERL-422 1

IUPAC Name:

Cycloaliphatic Epoxy Resin ERL-422 1

Specific details on test material used for the study:

- Name of test material (as cited in study report): ERL-4221

- Substance type: Epoxy resin
- Physical state: Clear liquid
- Analytical purity: Not documented

- Purity test date: Not documented
- Lot/batch No.: Not documented

- Stability under test conditions: Not documented
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc., Denver, Pennsylvania
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: Males: 338 to 390 grams
Females: 300 to 381 grams
- Housing: Individually in suspended, stainless steel cages with wire mesh bottoms.
- Diet (e.g. ad libitum): Agway Purina Guinea Pig Diet, ad libitum
- Water (e.g. ad libitum): Automatic watering system, ad libitum from the municipal water supply.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not documented but monitored and recorded twice daily
- Humidity (%): Not documented but monitored and recorded daily.
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark.

IN-LIFE DATES: From: 2 April 1991 To: 26 April 1991

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

propylene glycol

Induction 2

Concentration / amount:

Test material concentration used for intradermal injection induction phase - 5% v/v
Test material as supplied was not irritating and so undiluted (100%) ERL-4221 was applied topically in the induction phase to areas on the dorsum pre-treated with a mild irritant - sodium lauryl sulphate
100%, undiluted test material applied topically in the challenge phase

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

propylene glycol

Challenge 2

Concentration / amount:

Test material concentration used for intradermal injection induction phase - 5% v/v
Test material as supplied was not irritating and so undiluted (100%) ERL-4221 was applied topically in the induction phase to areas on the dorsum pre-treated with a mild irritant - sodium lauryl sulphate
100%, undiluted test material applied topically in the challenge phase

No. of animals per dose:

20 animals (10 male and 10 female) in the sensitisation assay with a further five males and five females treated as vehicle controls to assess background irritation. A further 4 males and 4 females were used in the preliminary investigation

Details on study design:

RANGE FINDING TESTS:
To confirm that the concentration proposed for intradermal injection (5.0%) did not produce extensive necrosis or ulceration or severe systemic toxicity, two animals were administered intradermal injections (2 sites per animal) of a 5.0% v/v concentration of the test material in propylene glycol. Injections of 0.1 ml per site were made intradermally. Observations were made at 24 and 48 hours for necrosis and ulceration.

Results indicated that a 5.0% concentration produced only local necrosis (i .e., no extensive necrosis or ulceration occurred) . Therefore, this concentration was used for the intradermal induction administration in the main experiment.

A topical range-finding study was perfomed using 6 animals (3 males and 3 females) to determine the lowest concentration which produced mild irritation and the highest concentration which did not produce irritation. Concentrations of 10, 25, 50 and 100 % were used.
Each test material mixture was applied to saturation (0.1 ml) to a 2x2 cm square of filter paper, which was then placed directly on the test site, a shaved area on dorsal and lateral surfaces of the test animals. Each animal was dosed with the four different concentrations, at four different sites (one concentration/site) , two on either side of the spinal column. The sites were then covered with plastic sheeting which was secured by wrapping the torso of each animal with an elastic adhesive bandage. After 24 hours the bandages, sheeting and patches were removed.

Observations for signs of dermal irritation (erythema, oedema and eschar formation) were made approximately 24 and 48 hours after removal of the patches. At each observation, all treated sites were scored for erythema, oedema and eschar formation.

MAIN STUDY
A. INDUCTION EXPOSURE
Each animal received two injections according to the following test concentrations:

Site one: Two sites with 0.1 ml of FCA/water emulsion per site.
Site two: Two sites with 0.1 ml of test material/Propylene glycol per site.
Site three: Two sites with 0.1 ml of test material/FCA/water emulsion per site.

The animals were injected intradermally on Day 0 of the testing schedule. On day seven, the animals were topically treated with the test substance. The hair in the shoulder area was re-clipped prior to topical application. As the test material was non-irritating at 100% concentration, the area was pre-treated with 10% sodium lauryl sulfate in petrolatum 24 hours before the test material was applied in order to provoke a mild inflammatory reaction.

The test material was applied to a 2 x 4 cm filter paper to saturation (approximately 0.2mls). The filter paper was then placed on the test site and secured with tape, which was then covered by overlapping impermeable plastic firmly secured by an elastic adhesive bandage wound around the torso of the animal.

In the range-finding test, the dorsal and lateral surfaces were used For the main experiment, prior to the injections, the hair in the shoulder region (approximately 4x6 cm) was clipped short with an electric clipper.

Control group: The control group (10 animals, 5 male and 5 female) were treated in the same way as the test animals, except they received polypropylene glycol or 70% ethanol instead of the test substance.

B. CHALLENGE EXPOSURE
On day 21, the hair was removed from a 5x5 cm area on the flank, by clipping. Patches were applied to the flank using the same procedure as for topical application on Day 7, except that a 2x2 cm piece of filter paper and approximately 0.1 mls of test material was used and remained on the animal for 24 hours. Dermal readings were made on all animals 24 and 48 hours after the removal of the patches. The challenge area was gently clipped after the 24-hour observation.

For the irritation control animals, ten (5 males and 5 females) previously untreated animals were subjected to the same challenge procedures as the animals which received the three induction exposures in order to differentiate dermal reactions produced by irritation from those produced by sensitization.

Challenge controls:

In order to differentiate dermal reactions produced by irritation from those produced by sensitization, ten (5 males and 5 females) previously untreated animals were subjected to the same challenge procedures as the animals which received the test material induction exposures.

Positive control substance(s):

yes

Remarks:

dinitrochlorobenzene. A positive control study was conducted circa evey 4-6 month to create a historical database, to ensure the study design and the strain of animals showed robust ability and sensitivity to detect potential sensitisers.

Study design: in vivo (LLNA)

Concentration:

Not applicable

No. of animals per dose:

Not applicable

Details on study design:

Not applicable

Statistics:

Not applicable

Results and discussion

Positive control results:

From the historical data presented by the testing laboratory, the positive control, dinitrochlorobenzene, was valid for use as 100% of the animals tested gave positive responses for sensitisation.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%, undiluted

No. with + reactions:

12

Total no. in group:

19

Clinical observations:

One animal died on Day 11. Twelve of the surviving test animals exhibited clear dermal responses (score of one or greater) at challenge with the remaining seven animals exhibiting scores of 0.5.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%, undiluted. No with. + reactions: 12.0. Total no. in groups: 19.0. Clinical observations: One animal died on Day 11. Twelve of the surviving test animals exhibited clear dermal responses (score of one or greater) at challenge with the remaining seven animals exhibiting scores of 0.5..

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%, undiluted

No. with + reactions:

8

Total no. in group:

19

Clinical observations:

One animal died on Day 11. Eight clearly positive reactions were evident and equivocal responses in a further seven animals. No reactions were observed for the controls at either time point

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%, undiluted. No with. + reactions: 8.0. Total no. in groups: 19.0. Clinical observations: One animal died on Day 11. Eight clearly positive reactions were evident and equivocal responses in a further seven animals. No reactions were observed for the controls at either time point.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Not applicable

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not applicable

Any other information on results incl. tables

One of the 10 sensitization group males was found dead on Day 11. Gross post-mortem results revealed discoloured lungs, the surface of the liver was yellow and the abdominal cavity was filled with yellow fluid, the causeof death was not established. As all remaining animals were free of any signs of toxicity, this death was probably not related to test material administration. All other animals survived throughout the study.

All surviving sensitization animals and all irritant control animals gained weight during the study. No remarkable results were noted during weekly physical examinations.

Incidence of Dermal Response at Challenge:

			Dermal Scores								Total No. of Animals
Group	Material	Time	0	0.5	1	2	3	Ed	N	E
IA	ERL-4221	24 48	0 4	7 7	11 8	1 0	0 0	5 0	0 0	0 0	19 19
IB	ERL-4221 (Irritation Control)	24 48	10 10	0 0	0 0	0 0	0 0	0 0	0 0	0 0	10 10

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information: Moderate Sensitiser

Conclusions:

Under the conditions of this study, ERL-4221 exhibited a potential to produce dermal contact sensitization in the guinea pig. The report author concludes that based on the 63% incidence of positive sensitization response, ERL-4221 would be categorized as a Moderate Sensitizer (Grade III). Re-evaluation of the results indicates a positive response was only confirmed in 8 of 19 animals after the 48 hour assessment and so the overall responder rate may be only 42% but this exceeds EU criteria for classification using the Magnusson and Kligman maximisation study method.

Based on the results of this study, the test substance would be classified as a skin sensitiser according to Regulation EC No. 1272/2008 and would have the following hazard statement associated with it: H317: May cause an allergic skin reaction. In addition, it should also be classified as Xi irritant and carry the risk phrase R43 in accordance with Directive 67/548/EEC.

Executive summary:

In a study conducted by Blaszcak (1991), the test substance, ERL-4221 was examined for its ability to act as a skin sensitiser when tested on Hartley Albino guinea pigs in a Guinea Pig Maximisation Test using the Magnusson-Kligmann method. A range-finding study was conducted to determine the appropriate concentration to use in the main study. This was determined to be 5%. 20 test animals were used (10 males and 10 females). They were intradermally induced on Day 0, then 7 days later the test substance was applied to the test animals topically. On day 21, the test animals were exposed to a challenge. Observations were recorded at 24 and 48 hours following termination of the challenge.

One male animal died on Day 11 of the experiment, 4 days after the 2nd induction. The remaining surviving animals exhibited clear dermal responses at challenge, with scores of 0.5 and 1.

Under the conditions of this study, ERL-4221 exhibited a moderate potential to produce dermal sensitization in the guinea pig. Based on the 63% incidence of sensitization, ERL-4221 would be categorized as a Moderate Sensitizer (Grade III).

Based on the results of this study, the test substance would be classified as a skin sensitiser Category 1B according to Regulation EC No. 1272/2008 and would have the following hazard statement associated with it: H317: May cause an allergic skin reaction. In addition, it should also be classified as Xi irritant and carry the risk phrase R43 in accordance with Directive 67/548/EEC.