Tall oil, potassium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 January 1992 to 22 February 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Justification for type of information:

Study conducted before the requirement of LLNA study was implemented.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An OECD 406 test was already conducted so it was considered unethical to run an LLNA test for animal welfare reasons.

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil

EC Number:

232-304-6

EC Name:

Tall oil

Cas Number:

8002-26-4

Molecular formula:

Not applicable as it is a UVCB

IUPAC Name:

Distilled Tall oil

Test material form:

other: liquid (unspecified)

Details on test material:

- Appearance: yellowish-brown liquid
- Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 6 to 7 weeks on arrival.
- Weight at study initiation: 301 to 370 g on arrival.
- Housing: The guinea-pigs were housed in groups of ten in suspended metal cages with wire mesh floors.
- Diet (e.g. ad libitum): A vitamin C enriched guinea-pig diet was provided ad libitum. Hay was given weekly.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 21 °C.
- Humidity (%): 30 to 70 % relative humidity.
- Air changes (per hr): Air exchange was maintained at approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light (0700 to 1900 hours) in each 24 hour period.

IN-LIFE DATES: From: 28 January 1992 To: 22 February 1992

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: Intradermal and topical

Vehicle:

other: Alembicol D

Induction 2

Concentration / amount:

Induction intradermal injection - 7.5 %
Induction topical application - as supplied
Topical challenge - as supplied and 50 % v/v

Challenge

Challenge 1

Route:

other: topical

Vehicle:

other: Alembicol D

Challenge 2

Concentration / amount:

Induction intradermal injection - 7.5 %
Induction topical application - as supplied
Topical challenge - as supplied and 50 % v/v

No. of animals per dose:

20 animals were used for the test group.

Details on study design:

RANGE FINDING TESTS: Yes. Animals were exposed to the test material in the intradermal induction at concentrations of 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 % v/v. For the topical induction, the animals were exposed to the test material at concentrations of 50, 60 and 75 % v/v and as supplied.

MAIN STUDY
A. INDUCTION EXPOSURE
INTRADERMAL INJECTIONS
A 40 x 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made into a 2 x 4 cm area within the clipped area as follows:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation (Ph.Eur.).
2. Test material 7.5 % v/v in Alembicol D.
3. Test material 7.5 % v/v in a 50:50 mixture of Freund's complete adjuvant and Alembicol D.
TOPICAL APPLICATION
The preliminary investigation indicated that the maximum practical concentration of the test material for topical application (as supplied) did not produce skin irritation. Therefore, six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.2 mL per site of 10 % w/w sodium lauryl sulphate in petrolatum. Twenty-four hours later, a 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 mL of the test material, as supplied. 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. The dressing was left in place for 48 hours.

In the induction phase, the control animals were treated similarly with the exception that the test material was omitted.

B. CHALLENGE EXPOSURE
The control and test animals were challenged topically two weeks after the topical induction application using the test material as supplied and 50 % v/v in Alembicol D.
Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 mL of the test material as supplied and applied to an anterior site on the flank. The test material 50 % v/v in Alembicol D was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".
The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.

OBSERVATIONS
- 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 of observation of dermal responses in the challenge.
- Dermal responses: The dermal reactions resulting from intradermal injection and topical application on the preliminary study, and topical application at the challenge were assessed using the following numerical system.

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

Any other lesion not covered by this scoring system was described.

INTERPRETATION OF RESULTS
Dermal reactions in the test animals elicited by the challenge application were compared with the findings simultaneously obtained in the control animals.
A test animal was considered to show positive evidence of delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals of the control group.
If the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result for that test animal was classified as inconclusive.
A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than the maximum reaction seen in animals of the control group.

Positive control substance(s):

yes

Remarks:

Formalin

Results and discussion

Positive control results:

The sensitivity of the guinea-pig strain used at the testing facility was checked periodically with formalin. Formalin was shown to cause sensitisation and therefore demonstrate the suitability of the test system.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

Test material as supplied and 50 % v/v in Alembicol D

No. with + reactions:

0

Total no. in group:

20

Results 2

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

Test material as supplied and 50 % v/v in Alembicol D

No. with + reactions:

0

Total no. in group:

20

Results 3

Key result

Reading:

rechallenge

Hours after challenge:

72

Group:

test chemical

Dose level:

Test material as supplied and 50 % v/v in Alembicol D

No. with + reactions:

0

Total no. in group:

20

Results 4

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

20

Results 5

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

20

Results 6

Key result

Reading:

rechallenge

Hours after challenge:

72

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

20

Results 7

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

Formalin

No. with + reactions:

20

Total no. in group:

20

Results 8

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

Formalin

No. with + reactions:

20

Total no. in group:

20

Results 9

Key result

Reading:

rechallenge

Hours after challenge:

72

Group:

positive control

Dose level:

Formalin

No. with + reactions:

20

Total no. in group:

20

Any other information on results incl. tables

Clinical Signs

No signs of ill health or toxicity were recorded.

 

Bodyweight

Bodyweight increases were recorded for all guinea-pigs over the period of the study.

 

Induction

- Intradermal injections: Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Irritation was seen in test animals at sites receiving the test material 7.5 % v/v in Alembicol D and slight irritation was observed in control animals receiving Alembicol D.

- Topical application: Slight erythema was observed in test animals following topical application with the test material as supplied. Very slight erythema was seen in the control guinea-pigs.

 

Challenge

The dermal responses seen in the test animals were similar to those of the controls.

Table 1 Dermal Reactions Observed After Induction

Site	Intradermal injection		Topical Application
	Test Animals	Control Animals	Test Animals	Control Animals
1	Necrosis	Necrosis	Slight erythema	Very Slight erythema
2	Irritation	Slight irritation
3	Necrosis	Necrosis

 

Table 2 Dermal Reactions Observed After the Challenge Application in Control Animals

Guinea-Pig Number		Score						Guinea-Pig Number		Score
		24 Hours		48 Hours		72 Hours				24 Hours		48 Hours		72 Hours
		A	P	A	P	A	P			A	P	A	P	A	P
200	E O	0 0	0 0	0 0	0 0	0 0	0 0	210	E O	0 0	0 0	0 0	0 0	0 0	0 0
201	E O	0 0	0 0	0* 0	0 0	0* 0	0 0	211	E O	0 0	0 0	0 0	0 0	0 0	0 0
202	E O	0 0	0 0	0* 0	0 0	0* 0	0 0	212	E O	0 0	0 0	0 0	0 0	0 0	0 0
203	E O	0 0	0 0	0* 0	0 0	0* 0	0 0	213	E O	L1 0	0 0	0 0	0 0	0 0	0 0
204	E O	0 0	0 0	0 0	0 0	0 0	0 0	214	E O	0 0	0 0	0 0	0 0	0 0	0 0
205	E O	0 0	0 0	0* 0	0 0	0* 0	0 0	215	E O	0 0	0 0	0 0	0 0	0 0	0 0
206	E O	0 0	0 0	0 0	0 0	0 0	0 0	216	E O	0 0	0 0	0 0	0 0	0 0	0 0
207	E O	L1* 0	0 0	L1* 0	0 0	0* 0	0 0	217	E O	0 0	0 0	0* 0	0 0	0* 0	0 0
208	E O	0 0	0 0	0* 0	0 0	0* 0	0 0	218	E O	0 0	0 0	0 0	0 0	0 0	0 0
209	E O	0 0	0 0	0 0	0 0	0 0	0 0	219	E O	0 0	0 0	0 0	0 0	0 0	0 0

E = Erythema

O = Oedema

L = Localised dermal reaction restricted to a small area of the challenge site.

*= Dryness and sloughing of the epidermis.

A = Anterior site, exposed to the test material as supplied.

P = Posterior site, exposed to the test material 50 % v/v in Alembicol D

 

Table 3 Dermal Reactions Observed After the Challenge Application in Test Animals

Guinea-Pig Number		Score						Result	Guinea-Pig Number		Score						Result
		24 Hours		48 Hours		72 Hours					24 Hours		48 Hours		72 Hours
		A	P	A	P	A	P				A	P	A	P	A	P
220	E O	0 0	0 0	0* 0	0 0	0* 0	0 0	-	230	E O	0 0	0 0	0 0	0 0	0 0	0 0	-
221	E O	0 0	0 0	0 0	0 0	0 0	0 0	-	231	E O	0 0	0 0	0 0	0 0	0 0	0 0	-
222	E O	0 0	0 0	0 0	0 0	0 0	0 0	-	232	E O	0 0	0 0	0 0	0 0	0 0	0 0	-
223	E O	0 0	0 0	0 0	0 0	0 0	0 0	-	233	E O	0 0	0 0	0 0	0 0	0 0	0 0	-
224	E O	0 0	0 0	0 0	0 0	0 0	0 0	-	234	E O	0* 0	0 0	0* 0	0 0	0* 0	0 0	-
225	E O	0 0	0 0	0 0	0 0	0 0	0 0	-	235	E O	0 0	0 0	0 0	0 0	0 0	0 0	-
226	E O	0 0	0 0	0* 0	0 0	0* 0	0 0	-	236	E O	0 0	0 0	0 0	0 0	0 0	0 0	-
227	E O	0 0	0 0	0 0	0 0	0 0	0 0	-	237	E O	0 0	0 0	0 0	0 0	0 0	0 0	-
228	E O	0 0	0 0	0* 0	0 0	0* 0	0 0	-	238	E O	L1 0	0 0	0* 0	0 0	0* 0	0 0	-
229	E O	L1* 0	0 0	0* 0	0* 0	0* 0	0 0	-	239	E O	0 0	0 0	0 0	0 0	0 0	0 0	-

E = Erythema

O = Oedema

L = Localised dermal reaction restricted to a small area of the challenge site.

*= Dryness and sloughing of the epidermis.

A = Anterior site, exposed to the test material as supplied.

P = Posterior site, exposed to the test material 50 % v/v in Alembicol D

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Conclusions:

Under the conditions of this study, the test material did not produce evidence of skin sensitisation and therefore requires no classification in accordance with EU criteria.

Executive summary:

The potential of the test substance to cause skin sensitisation (delayed contact hypersensitivity) was investigated in a guinea-pig maximisation test conducted in accordance with the standardised guideline OECD 406 under GLP conditions. Twenty albino Dunkin-Hartley guinea-pigs were exposed to the test material. The concentrations used in the induction phase of the study were 7.5 % v/v in Alembicol D for the intradermal application and undiluted test material for the topical application. Formalin was used as the positive control substance. In the challenge application, the animals were exposed to the test material as supplied and 50 % v/v in Alembicol D. No signs of ill health or toxicity were observed throughout the study. The test material elicited dermal reactions similar to those seen in the control animals. Under the conditions of this study, the test material did not produce evidence of skin sensitisation (Parcell, 1992).