2-Oxetanone, 3-C14-16-alkyl 4-C15-17-alkylidene derivs.

Endpoint summary

Description of key information

In several maximisation tests the substance proved to be non-sensitizing.

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:

29 April 2002 - 30 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to internationally accepted guidelines and under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

other: Japan MAFF Testing Guideline of 12 Nosan No. 8147 (as this is in line with OECD 406.)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study performed prior to REACH

Specific details on test material used for the study:

Name of test substance: Basoplast 20 konz. K
Test substance No.: 01/0711-1
Batch No.: PE 251001 / BB Nr. 2/intern 34
Date of manufacturing: October 25, 2001
Physical state / appearance: Solid / beige
Degree of purity / content: 80.7 g/100 g (analytical report No.: 01L00590).
Homogeneity: The test substance was homogeneous by visual inspection.
Stability: The stability under storage conditions over the study period was guaranteed by the sponsor. The information on stabilty of the test substance, which appropriately defines the test batch, is under the responsibility of the sponsor.
Storage conditions: Room temperature

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, FRG
- Age at study initiation: about 7 weeks
- Weight at study initiation: 316-411g
- Housing: 5 animals per cage. Stainless steel wire mesh cages with plastic-coated grating, minimum floor area: 2,000 cm2
- Diet (e.g. ad libitum): Kuba Labordiät (Kaninchen/ Meerschweinchen-Haltungsdiät) Provimi Kuba SA, Kaiseraugst, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 29 April 2002 - 30 September 2002

Route:

intradermal and epicutaneous

Vehicle:

olive oil

Concentration / amount:

Range finding:
The test substance concentrations for the main test were selected based on a study with a comparable test substance performed by a consulting institute (intradermal and epicutaneous induction only).
topical application: 75% or 50% in olive oil.

Main study:
Induction:
Intradermal injection - test substance 1% in olive oil Ph.Eur./DAB or 1% in Freund's adjuvant / 0.9% aqueous NaCI-solution (1:1)
Topical - 75% in olive oil

Challenge:
Topical - 75% in olive oil

Route:

epicutaneous, occlusive

Vehicle:

olive oil

Concentration / amount:

Range finding:
The test substance concentrations for the main test were selected based on a study with a comparable test substance performed by a consulting institute (intradermal and epicutaneous induction only).
topical application: 75% or 50% in olive oil.

Main study:
Induction:
Intradermal injection - test substance 1% in olive oil Ph.Eur./DAB or 1% in Freund's adjuvant / 0.9% aqueous NaCI-solution (1:1)
Topical - 75% in olive oil

Challenge:
Topical - 75% in olive oil

No. of animals per dose:

5 control animals and 10 test animals.

A second control group (control group 2, 5 animals) has been intended for a potential 2nd challenge in case of borderline results at the 1st challenge. Control group 1 cannot be used in this case due to a possible sensitization by the single application of the test substance at the 1st challenge.

Details on study design:

RANGE FINDING TESTS:
The test substance concentrations for the main test were selected based on a study with a comparable test substance performed by a consulting institute (intradermal and epicutaneous induction only).

For the challenge the maximum non-irritant concentration was determined with the pretest using 3 female animals. For detecting a possible influence on irritating effects of previous intradermal treatment with Freund's adjuvant, animals pretreated with Freund's adjuvant / 0.9% aqueous NaCI-solution (1: 1) each, in the same manner as intradermal induction for the test group referring front row (A) and back row (C) without test substance 2
weeks prior to the application of the test substance were used.

The test substance was applied to the skin of the flanks for 24 hours under occlusive dressing.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: single
- Exposure period: injections are given on day 0 and 6 days later the animals were topically exposed for 48 hours
- Test groups: 10
- Control group: two groups of 5 animals
- Site: neck region
- Frequency of applications: three pairs of intradermal injections and a topical application
- Duration: in total 8 days
- Concentrations: Intradermal injections: 1% test substance, Topical: 75% in olive oil

B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day(s) of challenge: 14 days after end of the induction period
- Exposure period: 24 hours
- Test groups: 20
- Control group: two groups of 5 animals
- Site: intact flank
- Concentrations: 75% in olive oil.
The test group and control group 1 were treated with the test substance formulation. Additionally, olive oil Ph.Eur./DAB was applied as a vehicle control. Control group 2 only received olive oil Ph.Eur./DAB.
- Evaluation (hr after challenge): 24 and 48 hours after removal of the patches

OTHER:
Weight check of the individual animals:
On day 0 and at the last day of observation.

A check for any dead or moribund animal was made twice each workday and once on Saturdays, Sundays and on public holidays.

The evaluation of the skin reactions was performed according to the following grading scale of Magnusson and Kligman:
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Description of any dermal findings Onot covered by this scale were recorded using the following abbreviations:
E = swelling
K = incrustation, partially open

Challenge controls:

Treated the same as the test animals.

Positive control substance(s):

yes

Remarks:

A separate study is performed twice a year in the Iaboratory. The positive controls with Alpha-Hexylcinnamaldehyde techn. 85% showed that the test system was able to detect sensitizing compounds under the Iaboratory conditions chosen.

Reading:

1st reading

Hours after challenge:

24

Group:

other: 1st control group

Dose level:

75% in olive oil (right flank) or just olive oil (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: 1st control group. Dose level: 75% in olive oil (right flank) or just olive oil (left flank). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Reading:

2nd reading

Hours after challenge:

48

Group:

other: 1st control group

Dose level:

75% in olive oil (right flank) or just olive oil (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: 1st control group. Dose level: 75% in olive oil (right flank) or just olive oil (left flank). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

24

Group:

other: 2nd control group

Dose level:

olive oil (left flank)

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: 2nd control group. Dose level: olive oil (left flank). No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: None.

Reading:

2nd reading

Hours after challenge:

48

Group:

other: 2nd control group

Dose level:

olive oil (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: 2nd control group. Dose level: olive oil (left flank). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75% in olive oil (right flank)

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75% in olive oil (right flank) . No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: None.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75% in olive oil (right flank)

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75% in olive oil (right flank) . No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

olive oil (left flank)

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: olive oil (left flank). No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: None.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

just olive oil (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: just olive oil (left flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Induction

After the intradermal induction intense erythema and swelling were observed at the injection sites of all control group animais and all test group animais at which only Freunds adjuvant/0.9% aqueous NaCI-solution (1: 1) was applied.

At the injection sites of a 1% test substance preparation in Freund's adjuvant/0.9% aqueous NaCI-solution (1: 1) intense erythema and swelling were seen in all test group animals.

lnjections of a 1% test substance preparation in olive oil Ph.Eur./DAB caused moderate and confluent erythema and swelling in all test group animals.

The control group animals, injected with olive oil Ph.Eur/DAB showed moderate and confluent erythema and swelling.

A 50% formulation of olive oil Ph.Eur./DAB with Freund's adjuvanh/0.9% aqueous NaCl-solution (1: 1) caused intense erythema and swelling in all control group animals. The epicutaneous induction with a 75% test substance preparation in olive oil Ph.Eur./DAB led to incrustation, partially open (caused by the intradermal induction) in addition to moderate and confluent erythema and swelling in 9 test group animals and intense erythema and swelling in one test group animal. The control group animals, which were applied with the vehicle, showed incrustation, partially open (caused by the intradermal induction) in addition to moderate and confluent erythema and swelling.

Challenge

The challenge with a 75% test substance preparation in olive oil Ph.Eur./DAB caused the following skin reactions:

Reading at 24 hours after removal of the patches:

- no skin findings in all animals of control group 1

- no skin findings in 8 out of 10 test group animais

- moderate and confluent erythema in 2 test group animals

Reading at 48 hours after removal of the patches:

- no skin findings in all animals of control group 1

- no skin findings in 8 out of 10 test group animais

- moderate and confluent erythema in 2 test group animals

Olive oil Ph.Eur./DAB which was applied as vehicle control to all control group and all test group animals caused discrete or patchy erythema in 1 out of 5 control group 2 animals (24 hours after removal of the patch) and in 1 out of 10 test group animals (24 and 48 hours after removal of the patches). No skin flndings were observed at the vehicle site of all animals of control group 1. Since no borderline results were observed, a 2nd challenge was not performed.

Body weights

The expected body weight gain was generally observed in the course of the study.

Tables with results:

1^stcontrol group	Right flank posterior: Form of application: Test substance 75%		Left flank posterior: Form of application: Olive 011 Ph./Eur. DAB in olive 011 Ph./Eur. DAB
Animal No.	24h	48h	24h	48h
311	0	0	0	0
312	0	0	0	0
313	0	0	0	0
314	0	0	0	0
315	0	0	0	0

Removal of the test substance (right flank posterior): with water

 

2^ndcontrol group	Left flank posterior: Form of application: Olive 011 Ph./Eur. DAB in olive 011 Ph./Eur. DAB
Animal No.	24h	48h
321	0	0
322	1	0
323	0	0
324	0	0
325	0	0

 

Test group	Right flank posterior: Form of application: Test substance 75%		Left flank posterior: Form of application: Olive 011 Ph./Eur. DAB in olive 011 Ph./Eur. DAB
Animal No.	24h	48h	24h	48h
331	0	0	0	0
332	0	0	0	0
333	0	0	0	0
334	2	2	1	1
335	0	0	0	0
336	0	0	0	0
337	0	0	0	0
338	0	0	0	0
339	0	0	0	0
340	2	2	0	0

Removal of the test substance (right flank posterior): with water

 

Interpretation of results:

GHS criteria not met

Conclusions:

The results of this study show that Basopast 20 konz. K does not have a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen.

Executive summary:

Basoplast 20 konz. K 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 a study with a comparable test substance performed by a consulting institute resp. on the results of a pretest. The intradermal induction was performed with a 1 % test substance preparation in olive oil Ph.Eur./DAB or 1 % 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 olive oil Ph.Eur./DAB. For the challenge a 75% test substance preparation in olive oil Ph.Eur./DAB 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 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 or intense erythema and swelling in all test group animals. After the challenge application moderate and confluent erythema was observed in 2 test

group animals at the test substance application sites. One of these animais also showed discrete or patchy erythema after application of the vehicle. Furthermore olive oil Ph.Eur.IDAB caused discrete or patchy erythema in 1 control group 2 animal.

The number of animais with skin findings after the challenge is summarized in the following table:

	Challenge
	Test substance 75% in olive oil Ph.Eur./DAB			Vehicle control: olive oh Ph.Eur./DAB
	24h	48h	Total	24h	48h	total
Control group 1	0/5	0/5	0/5	0/5	0/5	0/5
Control group 2*	No application of test substance			1/5	0/5	1/5
Test group	2/10	2/10	2/10	1/10	1/10	1/10

x/y: number of positive reactions/number of animals tested (reading at 24 h and/or 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.

It was concluded that Basopast 20 konz. K does not have a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are several studies available regarding the sensitisation properties of AKDs (see Table below).

The Alkyl Ketene Dimers manufactured and put on the market today is of superior quality as compared to the Ketene Dimer produced and marketed by Albright & Wilson in 1988. Since 1988 the manufacturing process of Albright and Wilson has improved, resulting in 96-98% completion of the process reactions. The improvement of the manufacturing process resulted in much lower content of impurities from raw material residuals as compared to the product marketed in 1988, for instance phosphorous based impurities have been reduced by>90%. The manufacturing process of today allows production of AKD with phosphorous based impurities at a typical level of 0-200 ppm as compared to >2000 ppm in the past. There may exist some products on the market with exceptional levels of up to 1000 ppm of phosphorous based impurities.

 

Also the production process for Basoplast 20 konz. has been changed and the new product, Basoplast 20 konz. K (batch PE251001 at 75% in oil) was shown to be negative in a Guinea Pig Maximisation Test (GPMT) from 2002 (BASF, 2002). Previously, one batch (Basoplast 20 konz., batch 216) administered as a 5% aqueous emulsion in a GPMT and applied as hot (60°C) melted product to the skin was found to be positive (BASF, 1994). However, the formulated product, a 20% emulsion of Basoplast 20 konz., batch P224, was tested in a Bühler test and found to be negative (BASF, 1995).

 

As a follow-up to the positive GPMT of one batch, batches of four other major products on the market were tested in GPMTs at challenge concentrations of up to 75% in oil (Aquapel 291, Aquapel 364 (batch 12MW1313), Keywax SF100 and Raisares (batch No E133), – All the products are chemically identical to Aquapel 364 and fall within the scope of CAS Number84989-41-3): All turned out to be negative. Since the chemical composition of all tested products was virtually the same the positive result with the one batch must have been an artifact of the test conditions or caused by a potent major impurity.

 

The effect of the improved manufacturing process on the sensitizing properties of AKD is reflected by the comparison between the sensitization studies performed on the Ketene Dimer from Albright and Wilson, 1988 and the Basoplast 20 konz., batch 216 from BASF, 1994 when the results indicated that AKD could be a sensitizer, and from 1990 and 2002 respectively, when the AKDs from the same manufacturers after improvement of the process reactions resulted in products which are not sensitizing.

 

The Albright and Wilson Ketene Dimer report from 1988 and the BASF Basoplast 20 konz. report from 1994 which indicate that AKD is a sensitizer are the only ones of the reports listed below with outstanding results. Since the qualities of Albright and Wilson Ketene Dimer produced in 1988 and BASF Basoplast 20 konz. from 1994 are no longer available on the market, the results from these studies are of no relevance today and should therefore be disregarded. Eventually all AKD products which are currently produced were tested negative for sensitization in the GPMT.

 

In conclusion, the overview of sensitization reports covering a variety of AKD products available on the market today all designate AKD as a non sensitizer, and therefore AKD is also classified as not sensitizing.

 

 

Reference	Test	Result	Product	Form	Chain	Status
Experimental Toxicology and Ecology, 2002 (BASF)	406 (M&K Test)	not sensitizing	Basoplast 20 konz. K	wax	Linear C16-C18	Key
ToxLabs, 1998 (Eka)	406 (M&KTest)	not sensitizing	Keywax SF 100	wax	Linear C16-C18	Supporting
CTL, 1997a (Hercules)	406 (M&KTest)	not sensitizing	Aquapel 364	wax	Linear C16-C18	Supporting
CTL, 1997b (Hercules)	406 (M&KTest)	not sensitizing	Aquapel 291	wax	Linear C16-C18	Supporting
Dept. of Toxicology, 1995 (BASF)	406 (Buehler Test)	not sensitizing	20% aqueous Bazoplast 20 konz. emulsion	wax	Linear C16-C18	Supporting
Inveresk, 1990 (Albright and Wilson)	406 (M&K Test)	not sensitizing	A & W Ketene Dimer	wax	Linear C16-C18	Supporting
Inveresk, 1988 (Albright and Wilson)	406 (M&KTest)	sensitizing	A & W Ketene Dimer Product no longer on the market	wax	Linear C16-C18	Disregard result
Dept. of Toxicology, 1994  (BASF)	406 (M&KTest)	sensitizing	Basoplast 20 konz. Product no longer on the market	wax	Linear C16-C18	Disregard result

 

Respiratory sensitisation

Endpoint conclusion

Additional information:

There is no information available on the sensitisation of the respiratory tract. Since the substance is a wax exposure humans via the inhalation route is considered unlikely to occur.

Justification for classification or non-classification

Conclusive data is available for skin sensitisation. These data do not justify classification for this endpoint.

 

There is no information available on the sensitisation of the respiratory tract. Since the substance is a wax exposure humans via the inhalation route is considered unlikely to occur. This information plus the data from the skin sensitisation studies do not justify classification for this endpoint.