2,2-bis(hydroxymethyl)butanoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-01-14 to 1997-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with recognised guideline.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Testing was carried out previous to the REACH regulation (EC) No 1907/2006. REACH concludes all available test data should be considered and used to fulfil data endpoints before any addition testing is undertaken. This data is valid to fulfil this endpoint, as such in used in this case

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis(hydroxymethyl)butanoic acid

EC Number:

424-090-1

EC Name:

2,2-bis(hydroxymethyl)butanoic acid

Cas Number:

10097-02-6

Molecular formula:

C6H12O4

IUPAC Name:

2,2-bis(hydroxymethyl)butanoic acid

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): DMBA
- Physical state: White solid
- Analytical purity: 99 %
- Lot/batch No.: 5J01
- Date of receipt: 1996-10-09
- Expiration date of the lot/batch: Six months from arrival
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England.
- Age at arrival: Approximately 4-5 weeks
- Weight at arrival: 314-355 g
- Housing: Animals were housed in groups of five in suspended metal cages with wire mesh floors.
- Diet: Vitamin C enriched guinea-pig diet FD2, ad libitum; Hay was given weekly
- Water: Drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.5-21 °C
- Humidity: 39-54 %
- Air changes: Approximately 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: From: 1997-01-14 To: 1997-02-14

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: intradermal injections and topical application

Vehicle:

other: water for irrigation - intradermal injection; distilled water - topical application

Induction 2

Concentration / amount:

Preliminary test:
- Intradermal injection: 0.01, 0.025, 0.05, 0.1 and 1.0 % w/v
- Topical application: 30, 50, 60 and 80 % w/v

Main test:
Intradermal injection: 0.05 % w/v DMBA in water for irrigation
Topical application: 80 % w/v DMBA in distilled water
Challenge application:
Anterior site: 60 % w/v DMBA in distilled water
Middle site: 30 % w/v DMBA in distilled water
Posterior site: Control - distilled water

Challenge

Challenge 1

Route:

other: topical application

Vehicle:

other: water for irrigation - intradermal injection; distilled water - topical application

Challenge 2

Concentration / amount:

Preliminary test:
- Intradermal injection: 0.01, 0.025, 0.05, 0.1 and 1.0 % w/v
- Topical application: 30, 50, 60 and 80 % w/v

Main test:
Intradermal injection: 0.05 % w/v DMBA in water for irrigation
Topical application: 80 % w/v DMBA in distilled water
Challenge application:
Anterior site: 60 % w/v DMBA in distilled water
Middle site: 30 % w/v DMBA in distilled water
Posterior site: Control - distilled water

No. of animals per dose:

Preliminary test: 2 males for intradermal injections (5 doses and control/animal) and 4 males for topical application (4 doses/animal)
Main test:
- Test item: 20 males
- Control: 10 males

Details on study design:

PRELIMINARY TEST:
- The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.
- Animals for these investigations were pre-treated with an intradermal injection of Freund's complete adjuvant, 50:50 with water for irrigation (Ph.Eur.), approximately four weeks prior to the start of the preliminary investigations.
- See attached document for information on tables of results for further details.

MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: One
- Exposure period: One week
- Test groups: On Day 1, three pairs of intradermal injections of 0.1 mL of Freund's complete adjuvant 50:50 with water for irrigation (Ph. Eur.), 0.05% w/v DMBA in water for irrigation, 0.05% w/v in a 50:50 mixture of water for irrigation and Freund's complete adjuvant, were made into a 20 x 40 mm area within the clipped area of dorsal skin on the scapular region.
- Control group: On Day 1, three pairs of intradermal injections of 0.1 mL of Freund's complete adjuvant 50:50 with water for irrigation (Ph. Eur.), water for irrigation, Freund's complete adjuvant 50:50 with water for irrigation, were made into a 20 x 40 mm area within the clipped area of dorsal skin on the scapular region.
- Site: Scapular region
- Frequency of applications: One
- Duration: One week

B. INDUCTION EXPOSURE: TOPICAL
- No. of exposures: One
- Day of exposures: Day 8
- Exposure period: 48 h
- Test groups: A 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 mL of compound, 60 % w/v in distilled water. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape (50 mm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage (50 mm width "Elastoplast") wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape.
- Control group: Control animals received the vehicle (distilled water) only and were treated similarly to the test animals.
- Site: Scapular region
- Frequency of applications: Single application
- Duration: Two weeks

C. CHALLENGE EXPOSURE: TOPICAL
- No. of exposures: One
- Day(s) of challenge: Day 22
- Exposure period: 24 h
- Test and control groups: A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 mL of DMBA, 60 % w/v in distilled water and applied to an anterior site on the flank. DMBA, 30 % w/v in distilled water was applied in a similar manner to a posterior site. A site on the right flank was treated with 0.2 mL of distilled water alone. The patches were sealed to the flank for 24 h under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".
- Evaluation (hr after challenge): 24 and 48 h after removal of the dressing.

OTHER:
- Clinical signs: All animals were observed daily for signs of ill health or toxicity.
- Bodyweight: The bodyweight of each guinea-pig on the main study was recorded on Day 1 (day of intradermal injections) and on the last day observations were made of dermal responses to the challenge applications.

Challenge controls:

Challenge control was topically applied with DMBA, 30 and 60 % w/v in distilled water.

Positive control substance(s):

yes

Remarks:

Hexyl cinnamic aldehyde, Benzocaine and Mercaptobenzothiazole from historical data

Results and discussion

Positive control results:

Hexyl cinnamic aldehyde, Benzocaine and Mercaptobenzothiazole induced sensitization reactions.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Anterior site: 60 % w/v DMBA in distilled water Middle site: 30 % w/v DMBA in distilled water Posterior site: Control - distilled water

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Inconclusive result in one animal

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Anterior site: 60 % w/v DMBA in distilled water Middle site: 30 % w/v DMBA in distilled water Posterior site: Control - distilled water. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Inconclusive result in one animal.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Anterior site: 60 % w/v DMBA in distilled water Middle site: 30 % w/v DMBA in distilled water Posterior site: Control - distilled water

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Inconclusive result in one animal

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Anterior site: 60 % w/v DMBA in distilled water Middle site: 30 % w/v DMBA in distilled water Posterior site: Control - distilled water. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Inconclusive result in one animal.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Anterior site: 60 % w/v DMBA in distilled water Middle site: 30 % w/v DMBA in distilled water Posterior site: Control - distilled water

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Anterior site: 60 % w/v DMBA in distilled water Middle site: 30 % w/v DMBA in distilled water Posterior site: Control - distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Anterior site: 60 % w/v DMBA in distilled water Middle site: 30 % w/v DMBA in distilled water Posterior site: Control - distilled water

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Anterior site: 60 % w/v DMBA in distilled water Middle site: 30 % w/v DMBA in distilled water Posterior site: Control - distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 5

Group:

positive control

Remarks on result:

not measured/tested

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under these test conditions, DMBA is not classified as sensitising to skin according to the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Executive summary:

Test Guidance

Magnusson & Kligman maximisation study (GPMT) performed according to EU Method B.6 (Skin Sensitisation) guideline and in compliance with GLP.

Method

Groups (20 males) of Dunkin-Hartley guinea pigs were induced with three pairs of intradermal injections of 0.1 mL of Freund's complete adjuvant 50:50 with water for irrigation (Ph. Eur.), 0.05% w/v DMBA in water for irrigation, 0.05% w/v in a 50:50 mixture of water for irrigation and Freund's complete adjuvant were performed on top, middle and down site of interscapular region, respectively. Control group (10 males) was intradermally induced with three pairs of 0.1 mL of Freund's complete adjuvant 50:50 with water for irrigation (Ph. Eur.), water for irrigation, Freund's complete adjuvant 50:50 with water for irrigation were performed on top, middle and down site of interscapular region, respectively. On Day 8, a 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 mL of test item, 60 % w/v in distilled water and the patch was placed over the intradermal injection sites for 48 h. Control animals received the vehicle (distilled water) only and were treated similarly to the test animals. After 2 weeks of rest period, a challenge application of test item dose formulations i.e., 0.2 mL of DMBA, 60 % w/v in distilled water and 30 % w/v in distilled water was applied to an anterior and posterior site on the flank, respectively and the right flank was treated with 0.2 mL of distilled water alone; the exposure period was 24 h. The test concentrations for the main study were determined from a preliminary toxicity study.

Results

No signs of ill-health or toxicity were observed. Intradermal injections produced necrosis at sites receiving Freund’s Complete Adjuvant in test and control animals. Slight irritation was seen in most test animals at sites receiving DMBA, 0.05 % w/v in water for irrigation and no irritation was observed in most control animals receiving water for irrigation. Well-defined to slight erythema with or without slight oedema was observed in control animals and test animals following topical application of DMBA, 80 % w/v in distilled water. Upon challenge application, there were no dermal reactions seen in 19/20 test animals or in any of the controls, therefore these 19 test animals gave negative responses and the remaining animal gave an inconclusive response. The positive controls (Hexyl cinnamic aldehyde, Benzocaine and Mercaptobenzothiazole) induced sensitization reactions indicating the validity of the study.

Conclusion

Under these test conditions, DMBA is not classified as sensitising to skin according to CLP Regulation (EC) N° (1272-2008).