3,5-dimethyl-1,2-dioxolane-3,5-diol

Endpoint summary

Description of key information

The guinea pig maximization test (OECD 406) showed that the test substance is a skin sensitiser. The data are conclusive, and the substance is classified as a Category 1b skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An apparently well conducted GLP study according to OECD 406 test guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

test commissioned before adoption of LLNA method

Species:

guinea pig

Strain:

Himalayan

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Supplied by BRL Limited, Basel, Switzerland.
- Age at study initiation: Approximately 4 - 9 weeks old.
- Body Weight: Approximately 500 gms.
- Housing: Five animals per metal cage with wire-mesh floors.
- Diet (e.g. ad libitum): standard guinea pig diet, including ascorbic acid (1000 mg/kg); (Charles River Breeding and Maintenance Diet for guinea pigs, Altromin, Lage, Germany.)
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 degrees Celcius
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route:

intradermal

Vehicle:

water

Concentration / amount:

Main study: Intradermal injection with a 5% concentration and epidermal challenge with a 100% concentration.

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

Main study: Intradermal injection with a 5% concentration and epidermal challenge with a 100% concentration.

No. of animals per dose:

10 females in the experimental group; 5 females in the control group.

Details on study design:

PRELIMINARY IRRITATION STUDY
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main Study. The selection of concentrations was based on the following criteria:

- The concentrations are well-tolerated by the animals.
- For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
- For challenge exposure: the maximum non-irritant concentration.

Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%,2%,1% and if needed, further lower concentrations using the same steps. The test system and procedures were identical to those used during the main study, unless otherwise specified. The five animals selected were between 4 and 9 weeks old and the body weights of some animals did exceed 500 grams. No body weights were determined at termination. .

Intradermal injections:
Initially, a series of four test substance concentrations was used; the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 ml/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hours after treatment. Based on the results in the initially treated animals, one additional animal was treated in a similar manner with two lower concentrations at a later stage.

Epidermal application:
A series of four test substance concentrations was used: the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 ml each) per animal to the clipped flank, using Metallline patches# (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape* and subsequently Caban elastic bandage*. The initially used animals receiving intradermal injections were treated with the lowest concentrations and two further animals' with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance. The resulting dermal reactions were assessed for irritation 24 and 48 hours after exposure.
* Suppliers: Lohmann GmbH, Neuwied, Germany (Coban) and 3M, St. Paul, Minnesota, U.S.A. (Metalline and Mlcropore)

MAIN STUDY

INDUCTION - Experimental animals

Day 1 The scapular region was clipped and three pairs of intradermal injections (0.1 ml/site) were made In this area as follows:
A) A 1:1 W/W mixture of Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 5% concentration.
C) A 1:1 wlw mixture of the test substance, at twice the concentration used in (6) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).

Day 3. The dermal reactions caused by the intradermal injections were assessed for irritation.

Day 7. The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SOS, Boom, Meppel, The Netherlands) in Vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

Day 8: The 10% SDS treated area between the injection sites was treated with 0.5 ml of a 100% test substance concentration using a Metalline patch (2x3 em) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance and the dermal reactions caused by the epidermal exposure were assessed for irritation.

INDUCTION - Control animals
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

CHALLENGE - All animals

Day 22: One flank of all animals was clipped and treated by epidermal application of a 100% test substance concentration and the vehicle (0.15 ml each), using Patch Test Plasters (Leukotest ®, Beiersdorf Medical, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Caban elastic bandage. The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

Challenge controls:

yes

Positive control substance(s):

no

Positive control results:

RESULTS

PRELIMINARY IRRITATION STUDY
Preliminary tests on intradermal injections and epidermal exposures were conducted for the selection of suitable test substance concentrations for the main study. Based on the results, the test substance concentrations selected for the Main Study were a 5% concentration for the intradermal induction and a 100% concentration for the epidermal induction exposure.

No signs of irritation were observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammalory reaction. A 100% test substance concentration was selecled for the challenge phase.

MAIN STUDY

Induction phase
The skin effects caused by the intradermal injections and epidermal exposure during the induction phase recorded. The reactions noted in the experimental animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

Challenge phase
Skin reactions of grade 4 were observed in all experimental animals in response to the 100% test substance concentration. No skin reactions were evident in the control animals. Eschar formation or scabs were seen in all treated skin sites among the experimental animals.

Toxicity I Mortality
One experimental animal was removed from the sludy on day 22 following the observation of piloerection, emaciation, watery discharge from the eyes, rales, dirty teeth and the animal felt cold. Macroscopic post-mortem examination showed scabs and/or alopecia on back and abdomen and pelvic dilation of the left kidney. It was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no further symptoms of systemic toxicity were observed in the remaining animals of the main study.

Group:

positive control

Remarks on result:

not measured/tested

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Normal

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.15 ml of 100% of the test substance

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

Eschar formation and scabs, Grade 4 (max)

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Normal

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.15 ml of 100% of the test substance

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

Eschar and scab formation, Grade 4 (max)

RESULTS

 

PRELIMINARY IRRITATION STUDY

Preliminary tests on intradermal injections and epidermal exposures were conducted for the selection of suitable test substance concentrations for the main study. Based on the results, the test substance concentrations selected for the Main Study were a 5% concentration for the intradermal induction and a 100% concentration for the epidermal induction exposure.

 

No signs of irritation were observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals wastreatedwith 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mildinflammaloryreaction. A 100% test substance concentration was selecled for the challenge phase.

 

MAIN STUDY

 

Induction phase

The skin effects caused by the intradermal injections and epidermal exposure during the induction phase recorded. The reactions noted in the experimental animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

 

Challenge phase

Skin reactions of grade 4 were observed in all experimental animals in response to the 100% test substance concentration. No skin reactions were evident in the control animals. Eschar formation or scabs were seen in all treated skin sites among the experimental animals.

 

ToxicityIMortality

One experimental animal was removed from thesludy on day 22 following the observation of piloerection, emaciation, watery discharge from the eyes, rales, dirty teeth and the animal felt cold. Macroscopic post-mortem examination showed scabs and/or alopecia on back and abdomen and pelvic dilation of the left kidney. It was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no further symptoms of systemic toxicity were observed in the remaining animals of the main study.

 

Body Weights

Body weights and body weight gain of experimental animals remained in the same range as controlsover the study period.

 

Grading Irritation Reactions:

Erythema and eschar formation:

No erythema .......................................................................................................................... 0

Slightly erythema (barely perceptible) ................................................................................ I

Well-defined .erythema .......................................................................................................... 2

Moderate erythema................................................................................................................ 3

Severe erythema (beet redness) to slight eschar formation (injuries in depth)........ 4

 

Oedema fonnatlon:

No oedama .............................................................................................................................0

Slighl oedema (barely perceptible) .................................................................................... 1

Well-defined oedema (edges: of area well-definedby definite raising)....................... 2

Moderate oedema (raised approximately1 millimeter) .................................................. 3

Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure)...... 4

•Intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis.

 

Grading Challenge Reactions:

No visible change.............................................................. ................................................... 0

Discrete or patchy erythema................................................................................................ 1

Moderate and confluent erythema...................................................................................... 2

Moderate erythema and swellIng................................................................................ , .... 3

Intense erythema and swelling........................................................................................... 4

After the end of the study all animals were killed by asphyxiation using an oxygen/carbon dioxide procedure.

 

INTERPRETATION

 

The results for the experimental animals at the challenge phase were compared with the results for the control animals. Positive skin reactions (grade 1 or more) were considered signs of sensitlsation, provided that such reactions were not observed or were less persistent in the control group. A sensitisation rate (%) was calculated as follows: the number of sensitised animals as a proportion of the total number of animals in the experimental group.

CONCLUSION

The skin reaction observed in response to a 100% test substance concentration in all (of the 9) experimental animals in the challenge phasewere considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 100 per cent.

 

Interpretation of results:

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

Conclusions:

The skin reactions observed in response to a 100% test substance concentration in all (of the nine) experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 100 per cent.

Main study: Intradermal injection with a 5% concentration and epidermal challenge with a 100% concentration.

Based on CLP Annex I Section 3.4.2.2.3.2. and Section 3.4.2.2.3.3. if the intradermal induction concentration is >1 % and the incidence of sensitized guinea pigs is >30 % the potency is moderate and the resulting sub category is 1b.

Executive summary:

SUMMARY: Assessment for contact Hypersensitivity to trigonox 44B in the Albino Guinea Pig (Maximization Test).

The study was carried out based on the guidelines described in: EC Commission Directive 96154/EC,Part B.6, 'Skin Sensitisation" and OECD No. 406, "Skin Sensitisation', and based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens '.

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, ten experimental animals were intradermally injected with a 5% concentration and epidermally exposed to a 100% concentration. Five control animals were similarly treated, but with vehicle alone (water). Approximately 24 hours before the epidermal induction exposure all animals were treated with 1 0% SDS. Two weeks after the epidermal application the animals were challenged with a 100% test substance concentration or the vehicle.

In the challenge phase, skin reactions of grade 4 were observed in all (01 nine) experimental animals in response to the 100% test substance concentration. No skin reactions were evident in the control animals. Eschar formation or scabs were seen in all treated skin sites among the experimental animals. One experimental animal was removed from the study on day 22 following the observation of piloerection, emaciation, watery discharge from the eyes, rales, dirty teeth and the animal felt cold. Macroscopic post-mortem examination showed scabs and/or alopecia on back and abdomen and pelvic dilation of the left kidney.

It was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no further symptoms of systemic toxicity were observed in the remaining animals of the main study. The skin reactions observed in response to a 100% test substance concentration in all (of the nine) experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 100 per cent.

Based on CLP Annex I Section 3.4.2.2.3.2. and Section 3.4.2.2.3.3. if the intradermal induction concentration is >1 % and the incidence of sensitized guinea pigs is >=30 % the potency is moderate and the resulting sub category is 1A.

 

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In the main study of the guinea pig maximization test, ten experimental animals were intradermally injected with a 5% concentration and epidermally exposed to a 100% concentration. Five control animals were similarly treated, but with vehicle alone (water). Approximately 24 hours before the epidermal induction exposure all animals were treated with 1 0% SDS. Two weeks after the epidermal application the animals were challenged with a 100% test substance concentration or the vehicle. In the challenge phase, skin reactions of grade 4 were observed in all (01 nine) experimental animals in response to the 100% test substance concentration. The skin reactions observed in response to a 100% test substance concentration in all (nine) experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals.These results indicate a sensitisation rate of 100 per cent. Based on these results and according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Guidelines in·Commission Directive93/21/EEC), the test substance should be labelled as: may cause sensitisation by skin contact (R43). 

This is a well conducted study.

Migrated from Short description of key information:
Guinea Pig (Maximisation Test): The study was carried out based on the OECD No. 406, "Skin SensHisation' guideline as prescribed by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens '. The skin reactions observed in response to a 100% test substance concentration in all (of the 9) animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals.These results indicate a sensitisation rate of 100 %.
 
In the main study, 10 experimental animals were intradermally injected with a 5% concentration and epidermally exposed to a 100% concentration. Five control animals were similarly treated, but with vehicle alone (water). Approximately 24 hours before the epidermal induction exposure all animals were treated with 1 0% SDS. Two weeks after the epidermal application the animals were challenged with a 100% test substance concentration and the vehicle. In the challenge phase, skin reactions of grade 4 were observed in all (01 nine) experimental animals in response to the 100% test substance concentration. No skin reactions were evident in the control animals. Eschar formation or scabs were seen in all treated skin sites among the experimental animals. One experimental animal was removed from the study on day 22 following the observation but upon post-mortem examination it was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no further symptoms of systemic toxicity were observed in the remaining animals of the main study. The skin reactions observed in response to a 100% test substance concentration in all (of the 9) experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals.These results indicate a sensitisation rate of 100%.

Justification for selection of skin sensitisation endpoint:
The only and a well-conducted study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The guinea pig maximization test conclusively showed that the test substance is a skin sensitiser. The data are conclusive, and the substance is classified as a Category 1b skin sensitiser ( H317).