2,2'-methylenebis(4,6-di-tert-butyl-phenyl)-2-ethylhexyl phosphite

Endpoint summary

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:

03 October 1995 to 27 October 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

In vivo data already available (study conducted in 1995)

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Species: Dunkin Hartley Crl: (HA)BR albino guinea pig (SPFquality). Recognised by international guidelines as the recommended test system (e.g. OECD, EEC).
Source: Charles River, Germany
Number of animals: Experimental group 10 females; Control group 5 females
Age at start of study: Approx. 5 weeks
Body weight prior to start: 362 - 423 grams
Identification: Ear tag
Test system check: A positive control experiment is performed once every six months as a sensitivity check of the test system.
ANIMAL HUSBANDRY
Conditions: Air-conditioned room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity.
Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.
Accommodation: Housing of 1 or 2 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands). The acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet: Free access to standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4mm (Hope farms, Woerden, The Netherlands). Certificates of analysis were examined and retained in the NOTOX archives. In addition, hay (B.M.I., Helmond, The Netherlands) was provided once a week.
Water: Free access to tap water, diluted with decalcified water. Certificates of analysis were examined and retained in the NOTOX archives.

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

Induction: The test substance at a 10% concentration (intradermal) and 50% test substance (epidermially)

Challenge: test substance concentrations, 50%, 25% and 10%

Route:

epicutaneous, semiocclusive

Vehicle:

corn oil

Concentration / amount:

Induction: The test substance at a 10% concentration (intradermal) and 50% test substance (epidermially)

Challenge: test substance concentrations, 50%, 25% and 10%

No. of animals per dose:

Control group: 5 animals
Experimental group: 10 animals

Details on study design:

PRELIMINARY IRRITATION STUDY
Prior to the start of the Main study, the intradermal and epidermal irritancy of ADK STAB HP-10 was investigated to select test substance concentrations suitable for the induction and challenge phase of the Main Study.
The test system, procedures and techniques were identical to those used during the main study, unless otherwise mentioned.
The animals were selected from stock and were between 5 and 9 weeks old.
Intradermal injections: A 10% and a 5% concentration were injected in one animal into two injection sites (0.1 ml each) in the right and left clipped shoulder regions, respectively. A 2% and 1% concentration were injected in a similar manner in a second animal. The resulting dermal reactions were assessed 24 and 48 hours later for erythema (see scale below), necrosis and diameter of necrosis.
Epidermal application: A 50% concentration (0.5 ml) was applied epidermally on a clipped flank of one animal, using a Scotchpak-non-woven patch (2.5 x 2.2 em) on Micropore tape and held in place by Caban elastic bandage*. A 25% concentration was applied in a similar manner on a second animal.
Four concentrations of the test substance (50%, 25%, 10% and 5%, 0.05 ml each) were applied occlusively on a clipped flank of each of two other animals using Square chambers (v.d. Bend, Brielle, The Netherlands), mounted on Micropore tape and held in place by Caban elastic bandage.
After 24 hours, the dressings were removed and the skin cleaned of residual test substance. The treated skin-sites were assessed 24 and 48 hours after exposure according to the scale described below.

Erythema and eschar formation:
No erythema 0
Slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Slight oedema (barely perceptible) 1
Well-defined oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more then millimetre and extending beyond the area of exposure) 4

MAIN STUDY
INDUCTION - EXPERIMENTAL ANIMALS
Day 1: Three pairs of intradermal injections (0.1 ml/site) were made in the clipped scapular region as follows:
A) Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.), 50:50 with water for injection (Ferensius AG, Bad Homburg, Germany).
B) The test substance at a 10% concentration.
C) The test substance, at twice the concentration used in (B), emulsified in a 50:50 mixture of Freunds' Complete Adjuvant.
Day 3: The dermal reactions caused by the intradermal injections were recorded.
Day 7: The clipped scapular area was rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.
Day 8: The clipped area between the injection sites was treated with 0.5 ml of a 50% test substance concentration using a Scotchpak-non-woven patch (2 x 4 cm) mounted on Micropore tape and secured with Caban elastic bandage.
Day 10: After 48 hours, the dressings were removed and the skin cleaned of residual test substance. Subsequently, all dermal reactions caused by the epidermal exposure were assessed according the scale presented before.

INDUCTION - CONTROL ANIMALS
Day 1: Intradermal injections were made similar to the method described above with the omission of the test substance.
Day 3: The dermal reactions caused by the intradermal injections were recorded.
Day 7: SDS 10% treatment, as described above.
Day 8: Epidermal treatment as described for the experimental animals, but with the vehicle only.
Day 10: After 48 hours, the dressings were removed and the skin cleaned. Subsequently, all dermal reactions caused by the epidermal exposure were assessed according the scale presented before.

CHALLENGE
Day 22: All animals were treated epidermally with 0.05 ml of each of a the following test substance concentrations, 50%, 25% and 10% and the vehicle on a clipped flank, using Square chambers, attached to Micropore tape and secured with Caban elastic bandage.
Day 23: After approximately 24 hours, the dressings were removed and the skin cleaned of residual test substance and vehicle. The treated sites were assessed for erythema and swelling 24 and 48 hours after removal of the dressings, using the numerical grading system below. After the first reading, the test sites were re-clipped.

No visible changes 0
Discrete or patchy erythema 1
Moderate and confluent erythema 2
Moderate erythema and swelling 3
Intense erythema and swelling 4

At the end of the study period all animals were killed by oxygen/carbon dioxide asphyxiation.

OBSERVATIONS
Mortality/Viability: Twice daily
Toxicity: At least once daily.
Body weights: Prior to start and at termination of the study.

Challenge controls:

The results for the experimental animals at the challenge application(s) were compared with the results for the control animals.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0, 10, 25 & 50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0, 10, 25 & 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0, 10, 25 & 50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0, 10, 25 & 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 1.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 1.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Group:

positive control

Remarks on result:

other: positive control not used

PRELIMINARY STUDY: Based on the results, the test substance concentrations for the Main Study were selected to be a 10% concentration for the intradermal induction and a 50% concentration for the epidermal induction exposure.

Since no signs of irritation were observed to the concentration selected for the epidermal induction, it was decided to treat all animals with 10% SDS approximately 24 hours before the epidermal induction.

A 50%, 25% and 10% concentration were selected for the challenge phase.

 

MAIN STUDY

Challenge phase: Skin reactions, consisting of grade 1, were observed in two experimental animals in response to one or three test substance concentrations 24 hours after exposure only.

No skin reactions were evident in the control animals.

Toxicity Symptoms/Mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Body Weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

TABLE 1:PRELIMINARY IRRITATION STUDY

No signs of systemic toxicity were observed in any of the animals.

INTRADERMAL INJECTIONS

Animal No.	Bodyweight^a(grams)	Concentration % (w/w)	SKIN READINGS AFTER 24/48 HRS			Bodyweight^b(gram)
			Erythema	Necrosis	Diameter (mm)
634	431	10*	1/0	no/no	-/-	453
		5	1/0	no/no	-/-
635	433	2	0/0	no/no	-/-	454
		1	0/0	no/no	-/-

 

*A 25% concentration did pass through the needle, but could not be injected into the animal.

 

EPIDERMAL APPLICATION USING SCOTCHPAK-NON-WOVEN PATCH

Animal No.	Bodyweight^a(gram)	Concentration % (w/w)	SKIN READINGS AFTER BANDAGE REMOVAL				Bodyweight^b(gram)
			After 24 hours		After 48 hours
			Erythema	Oedema	Erythema	Oedema
634	431	50	0	0	0	0	453
635	433	25	0	0	0	0	454

 

EPIDERMAL APPLICATION USING SQUARE CHAMBERS

Animal No.	Bodyweight^a(gram)	Concentration % (w/w)	SKIN READINGS AFTER BANDAGE REMOVAL				Bodyweight^b(gram)
			After 24 hours		After 48 hours
			Erythema	Oedema	Erythema	Oedema
632	425	50	0	0	0	0	449
		25	0	0	0	0
		10	0	0	0	0
		5	0	0	0	0
633	433	50	0	0	0	0	442
		25	0	0	0	0
		10	0	0	0	0
		5	0	0	0	0

 

^aBodyweight prior to treatment

^bBodyweight at termination

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The skin reactions observed in the two experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 20 per cent.
Applying the rating of allergenicity described by Kligman A.M. (1966) on the results obtained in this test, ADI< STAB HP-10 is considered to have mild sensitising properties.
Based on these results and according to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), ADI< STAB HP-10 cannot be classified and has no obligatory labelling requirement.

Executive summary:

Assessment for Contact Hypersensitivity to ADK STAB HP-10 in the Albino Guinea Pig (Maximization Test).

The study was carried out in accordance with the DECO Guideline No. 406, "Skin Sensitisation", the EEC Directive 92/69/EEC, Part B.6, "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 (in corn oil) 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 10% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with omission of the test substance.

Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50%, 25% and 10% test substance concentration and the vehicle.

 

In the challenge phase, skin reactions grade 1 were observed in two experimental animals in response to one or three test substance concentrations at the 24 reading only. No skin reactions were evident in the control animals.

 

The skin reactions observed in the two experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 20 per cent.

Applying the rating of allergenicity described by Kligman A.M. (1966) on the results obtained in this test, ADK STAB HP-10 is considered to have mild sensitising properties.

Based on these results and according to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), ADK STAB HP-10 cannot be classified and has no obligatory labelling requirement.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Assessment for Contact Hypersensitivity to ADK STAB HP-10 in the Albino Guinea Pig (Maximization Test).

Test substance concentrations (in corn oil) 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 10% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with omission of the test substance.

Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50%, 25% and 10% test substance concentration and the vehicle.

In the challenge phase, skin reactions grade 1 were observed in two experimental animals in response to one or three test substance concentrations at the 24 reading only. No skin reactions were evident in the control animals.

The skin reactions observed in the two experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 20 per cent.

Applying the rating of allergenicity described by Kligman A.M. (1966) on the results obtained in this test, ADK STAB HP-10 is considered to have mild sensitising properties.

Migrated from Short description of key information:

Not sensitizing in the guinea pig maximisation test.

Justification for selection of skin sensitisation endpoint:

Endpoint conclusiong dervied from study using EU Method B6 and OECD guideline 406.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the test results and according to Regulation (EC) No 1272/2008 for classification, labelling and packaging, the substance is not classified for skin sensitisation.