Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione

Endpoint summary

Description of key information

In a GLP skin sensitisation study on guinea pigs according to the OECD Guideline 406 the test substance was assigned the Category 1 (skin sensitising) based on GHS criteria.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2002 until April 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD. When this study was performed, the LLNA had just been developed.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0767
- Source: BASF AG
- Purity: aqueous formulation: active ingredient 49.5 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Was guaranteed for the study period.
- Stability of the test substance in the solvent: was not determined
- Homogeneity: The test substance was homogeneous by visual inspection but was not investigated by analysis as it was applied in solution.
- physical state: liquid

OTHER:
- Correctness of the concentration: was confirmed by analysis

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

Hsd Poc: DH (SPF)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, FRG
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: SPF
- Age at study initiation: about 6 - 7 weeks
- Weight at study initiation: 340 g - 379 g
- Housing: fully air-conditioned rooms with five animals per cage
- Diet: Kliba Labordiät, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: Seven days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE: 19.03.2002 - 19.04.2002

Route:

intradermal

Vehicle:

other:

Remarks:

0.9% aqueous NaCl-solution

Concentration / amount:

5% test substance in 0.9% aqueous NaCl-solution or in (1:1) Freund's adjuvant/0.9% aqueous NaCl-solution

Day(s)/duration:

Day 0

Adequacy of induction:

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

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

75% test substance preparation in doubly distilled water

Day(s)/duration:

Day 7

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

75% test substance in doubly distilled water

Day(s)/duration:

Day 21 for 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test group: 10
Control group: 5

Details on study design:

RANGE FINDING TESTS
INTRADERMAL PRETEST:
Six intradermal injections in groups of two per animal were applied to each animal

A) front row: Two injections each of 0.1 ml Freund's adjuvant (Supplier: DIFCO LABORATORIES, Detrolt/Michigan, USA) without test substance emulsified with 0.9% aqueous NaCl-solution in a ratio of 1:1
B) middle row: Two injections each of 0.1 ml of 5% test substance in 0.9% aqueous NaCl-solution
C) back row: Two injections each of 0.1 ml Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) with 5% test substance.
- Site of application: neck region
- No. of animals: Two per test substance concentration
- Reading: 24 h after the beginning of application

EPICUTANEOUS PRETEST.
2 x 2 cm gauze patches (6 Iayers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) Gontaining 0.5 ml of the undiluted test substance /75% of the test substance formulation were applied to the skin of the flanks under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (5 x 5 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdort AG.
- Duration of exposure: 24 h
- Site of application: flank
- Number of test animals: Three per test substance concentration
- Readings: 1 h, 24 h, and 48 h after removal of the patches

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Six intradermal injections in groups of two per animal were given
- Control group:
A) front row: Two injections each of 0.1 ml Freund's adjuvant (Supplier: DIFCO LABORATORIES, Detrolt/Michigan, USA) without test substance emulsified with 0.9% aqueous NaCl-solution in a ratio of 1:1
B) middle row: Two injections each of 0.1 ml of the undiluted vehicle
C) back row: Two injections each of 0.1 ml of a 50% formulation of the vehicle without test substance emulsified with Freund's adjuvant/0.9% aqueous NaCl-solution (1:1)
- Site of application: neck region
- Frequency of applications: once
- Reading: 24 h after the beginning of application
- Test groups:
Same as for intradermal pretest, see above.

- epicutaneous induction:
- was carried out one week after intradermal induction
2 x 4 cm gauze patches (6 Iayers surgical gauze Ph. Eur. from Lohmann GmbH & Co.KG) containing 75% of the test substance formulation were applied to the 5km of the flank region under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 6 cm from Russka) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
1 ml of the test substance formulation was applied to each animal.
The control animais were not treated since the distilled water used as formulating agent was not expected to influence the result of the study.
- Duration of exposure: 48 hours
- Site of application: neck region, same area as in the case of the previous intradermal application
- Readings: 48 hours after the beginning of application

B. CHALLENGE EXPOSURE
- Duration of exposure: 24 hours
- Exposure period: challenge was performed 14 days after the epicutaneous induction
- Test groups and control group: were treated with the test substance formulation (control group 2 remained untreated)
- Site of application: intact flank
- Concentrations: 0.5 ml of 75% of the test substance formulation was applied to each animal.
- Readings: 24 and 48 hours afte rthe removal of the patch
2 x 2 cm gauze patches (6 Iayers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test substance formulation were applied to the skin of the flank under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (5 x 5 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.

Challenge controls:

no

Positive control substance(s):

yes

Remarks:

Seperate study conducted twice a year

Positive control results:

A positive control (reliability check) with a known sensitizer is not included in this study. However, a separate study is performed twice a year in the Iaboratory. The positive controls with alpha-Hexyl cinnamic aldehyde techn. 85% showed that the test system was able to detect sensitizing compounds under the Iaboratory conditions chosen.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75% test substance preparation in doubly distilled water

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Details on local reactions given below

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75% test substance preparation in doubly distilled water

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

Details on local reactions given below

Remarks on result:

positive indication of skin sensitisation

Table 1: Results of the Challenge Test in treated group

	Right flank middle: Form of application: Test substance 75% in doubly distilled water
Animal number	Findings 24 hours after removal of the patch	Findings 48 hours after removal of the patch
831	2 S	2 S
832	2	1
833	2 S	2 S
834	3 S	3+
835	2	1
836	1	0
837	2	2 S
838	2 S	2 S
839	2	2 S
840	3	2 SS

 

Assessment of the skin reactions:

0 = no visible change

1 discrete or patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

S =scaling

SS = severe scaling

+ = eczematoid skin change

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the evaluation criteria cited under 3.6. the results of this study show that the test substance has a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen.

Executive summary:

The test substance was tested for its sensitizing effect on the skin of the guinea pig in the Maximization Test based on the method of Magnusson and Kligman.

The test substance concentrations for the main test were selected based on the results of the pretest. The intradermal induction was performed with a 5% test substance preparation in 0.9% aqueous NaCI-solution or a 5% test substance preparation in Freund's adjuvant 1 0.9% aqueous NaCI-solution (1: 1) and the epicutaneous induction with a 75% test substance preparation in doubly distilled water. For the challenge a 75% test substance preparation in doubly distilled water was chosen. The study was initiated with 2 control groups and 1 test group.

The intradermal induction was performed on day 0 and the epicutaneous induction on day7.

A challenge was carried out 14 days after the epicutaneous induction. The intradermal induction caused moderate and confluent erythema and swelling or intense erythema and swelling at the injection sites of the test substance preparation in all test group animals. After the epicutaneous induction incrustation, partially open (caused by the intradermal induction) could be observed in addition to moderate and confluent

erythema and swelling in all test group animais. After the challenge discrete or patchy to intense erythema and swelling were observed

in addition to eczematoid skin change, scaling and severe scaling in all animais of the test group. The number of animals with skin findings after the challenge is summarized in the following table:

	Challenge
	Test substance 75% in doubly distilled water
	24 h	48 h	Total
Control group 1	0/5	0/5	0/5
Control group 2*	No application of test substance
Test group	10/10	9/10	10/10

x/y: number of positive reactions/number of animais tested (reading at 24 h andlor 48 h after the removal of the patch)

*: control group 2 that had been intended for a potential 2nd challenge was not treated with the test substance, since a 2nd challenge was not necessary on the basis of the unambiguous results of the 1st challenge.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The test substance was tested for its sensitizing effect on the skin of the guinea pig in a GLP-study according to the OECD Guideline 406 (Skin sensitisation).

The test substance concentrations for the main test were selected based on the results of a pretest. The intradermal induction was performed with a 5% test substance preparation in 0.9% aqueous NaCI-solution or a 5% test substance preparation in

Freund's adjuvant / 0.9% aqueous NaCI-solution (1: 1) and the epicutaneous induction with a 75% test substance preparation in doubly distilled water. For the challenge a 75% test substance preparation in doubly distilled water was chosen.

The study was initiated with 2 control groups and 1 test group.

The intradermal induction was performed on day 0 and the epicutaneous induction on day 7.

A challenge was carried out 14 days after the epicutaneous induction.

The intradermal induction caused moderate and confluent erythema and swelling or intense erythema and swelling at the injection sites of the test substance preparation in all test group animals.

After the epicutaneous induction incrustation, partially open (caused by the intradermal induction) could be observed in addition to moderate and confluent erythema and swelling in all test group animals.

After the challenge discrete or patchy to intense erythema and swelling were observed in addition to eczematoid skin change, scaling and severe scaling in all animals of the test group.

Based on the results of this study and applying the evaluation criteria it was concluded that the test substance has a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen.

Several instances of sensitisation in humans exposed to low concentrations also indicate that the chemical is a sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance caused skin sensitization in GPMT in 10/10 animals after intradermal induction with 5%. Due to the high induction dose used, but also the response in 100% of the animals, these results are considered insufficient to assigne a subcategory for sensitization. Consequently, the substance is classified for skin sensitization cat. 1 according to Regulation (EC) No. 1272/2008.