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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13.03.1995-21.04.1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
This method is similar to that described in the Official Journal of the European Communities No. L383 A131 B.6. Acute Toxicity (Skin Sensitization), 29.12.1992 and OECD Guideline 406 for Testing of Chemicals (1993).
Deviations:
not specified
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed in 1995. At that date OECD 406 was a fully accepted study.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Composition
Estimated composition based on all analyses :-
44.7% (w/w) water
49.8% (w/w) inositol hexaphosphoric acid
5.5% (w/w) partially phosphorylated inositol (e.g. inositol tetraphosphoric acid)
Specific details on test material used for the study:
Phytic acid diluted with aqua to yield a 50% solution is the commonly available/supplied composition.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: physiological saline (Ringer)
Concentration / amount:
0.1%/ 0.1 ml
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: physiological saline (Ringer)
Concentration / amount:
5,0 % a.i. (w/v)
Day(s)/duration:
48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
2.5% a.i. (w/v)
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 (10 male/ 10 female) were randomly selected for the test group
10 (5 male/ 5 female) were randomly selected for the control group
Details on study design:
TEST SOLUTIONS:
A. INDUCTION PHASE
- Test solutions for injection:
1) A 1:1 mix of Freund`s Complete Adjuvant (FCA) with 0.9% (w/v) physiological saline (saline).
2) 0.1 % a.i. (w/v) phytic acid in saline.
3) 0.2 % a.i. (w/v) phytic acid in saline mixed 1:1 with FCA to give a final concentration of 0.1 % a.i. (w/v) of phytic acid.

- Test solution for occlusive patch
5.0% a.i. (w/v) phytic acid in saline.

- Control solutions (Injection)
1. 1:1 FCA mix. and saline.
2. saline.

B. CHALLENGE PHASE
- Test solution for occlusive patch
2.5% a.i. (w/v) phytic acid in saline.

TEST METHOD:
INDUCTION PHASE
Induction consisted of two stages, intradermal injection followed seven days later by
occluded patch application.
- Intradermal injection
The hair was clipped from a 2cm x 4cm area of skin in the dorsal shoulder area and three
pairs of intradermal injections (test solution 1-3 / control solutions 1-2-1) were made within the clipped area
- Occlusive Patch
Seven days after the injections the same 2cm x 4cm area was clipped and shaved. A 2cm x 4cm filter paper patch, attached by double-sided adhesive tape to a 4cm x 6cm piece of thin polythene, was saturated with 5.0% a.i. phytic acid in saline and placed over the shaved site. The patch was held in place for 48 hours by adhesive plaster wrapped around the trunk behind the forelimbs.
Control group received an occluded patch of the test solvent over the injection sites.
The occluded patch site was scored 24 hours after removal of the bandage
- Challenge
Fourteen days after the application of the induction patch the guinea pigs of both groups were challenged on the clipped and shaved flank by occluded patch. For each animal, an 8mm diameter filter paper patch in an llmm aluminium patch test cup was saturated with 2.5% a.i. phytic acid in saline and the patch applied to the shaved flank. The patch was held in place for 24 hours by adhesive plaster wound around the trunk. The treatment sites were examined for evidence of sensitization 24 and 48 hours after removal of the patches
Challenge controls:
10 control guinea pigs were selected with similar body weights to those of the test guinea pigs at that challenge.
The guinea pigs received two intradermal injections of 1:1 mixture of FCA and saline, two O.l ml injections of saline and two O.l ml injections of a 1:1 mixture of
FCA and saline in exactly the same way as the test animals.
This was followed seven days later by a 24 hour occluded patch of the test solvent over the injection sites. After both of the induction procedures the animals were assessed in exactly the same way as the test animals.
At challenge the guinea pigs were challenged with the test material in exactly the same way as the test animals.
Positive control substance(s):
yes
Remarks:
Hexyl Cinnamic Aldehyde (periodic tests are run at the laboratory)

Results and discussion

Positive control results:
As a check on the sensitivity of the method carried out in ESL, one positive control material was subjected to the normal M&K test procedure at intervals specified below.
Summary data for a period relating up to and including the study of phytic acid were: HEXYL CINNAMIC ALDEHYDE
Concentrations:
Induction injection 0.5% (v/v)
Induction application 50.0% (v/v)
Challenge application 10.0% (v/v)

Results of tests
Study number Date % positive
SM930149 06 April 1993 80%
SM930150 23 November 1993 80%
SM940210 09 May 1994 70%
SM940414 01 November 1994 70%

For study number SM930150 the following concentrations were used:
Concentrations :
Induction injection 1.0% (w/v)
Induction application75.0% (w/v)
Challenge application 5.0% (w/v)

For study number SM940210 and SM940414 the following concentrations were used:
Concentrations: Induction injection
Induction application
Challenge application
1.0% (w/v)
50.0% (w/v)
10.0% (w/v)

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2.5% a.i. (w/v)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2.5% a.i. (w/v)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2.5% a.i. (w/v)
No. with + reactions:
1
Total no. in group:
9
Remarks on result:
no indication of skin sensitisation
Remarks:
10 animals were selected for the control group but one animal was found dead after induction application phase
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2.5% a.i. /w/v)
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
no indication of skin sensitisation
Remarks:
10 animals were selected for the control group but one animal was found dead after induction application phase
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10% (v/v) Hexyl Cinnamic Aldehyde
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
postive control is run periodically at the lbaoratory. No detailed information on readings, no of animals etfc. is availabe

Any other information on results incl. tables

3.3 Preliminary irritation tests

It was not considered necessary to adjust the pH of the test material for the sensitization

test as the results of the preliminary irritation tests showed that the irritancy of phytic acid

was not significantly altered and the concentrations selected would be the same.

As a result of the preliminary irritation tests, 0.1 % a.i. was selected from the preliminary

irritation test to be the highest suitably irritant concentration for the intradermal injection

induction. From the preliminary occluded patch test, 5.0% a.i. was selected as it was the

highest suitably irritant concentration for the patch induction. 2.5% a.i was selected as

the highest non-irritant concentration for the challenge patch. The results are given in

Appendix 1.

3.4 Sensitization test

The test and control guinea pigs showed intense (grade 3) erythema reactions to the

induction injection and induction application treatments. The individual scores for the test

animals are given in Table 2 and the control animals in Table 3.

There was no evidence of sensitization reactions in any of the twenty test guinea pigs

challenged with 2.5 % phytic acid at challenge 1.

Control animal B9 showed a minor irritation reaction to the test material at the 24 hour

assessment, the rest of the control animals showed no response. Test animal All showed

a minor irritation reaction to the test material at the 24 hour assessment.

The results are summarised in Table 1. Individual animal data for the challenge treatment

is given in Table 4 for the test animals and Table 5 for the control animals.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, phytic acid was classified as a non-sensitiser
Executive summary:

The study following the principles of OECD 406 (1993) (Magnusson & Kligman Maximization test) was performed to determine the potential of phytic acid to induce skin sensitization reactions.

Sensitization was induced in guinea pigs by intradermal injections of both test substance and Freund Is Complete Adjuvant. Seven days later the induction process was supplemented by application of the test substance to the shoulder injection sites under occlusion for 48 hours. Fourteen days later the guinea pigs were challenged by a 24 hour occluded patch of the test substance, the treatment sites were then examined for evidence of sensitization 24 and 48 hours after removal of the challenge patch.

The test and control guinea pigs showed intense (grade 3) erythema reactions to the induction injection and induction application treatments.

There was no evidence of sensitization reactions in any of the twenty test guinea pigs challenged with 2.5 % phytic acid at challenge 1.

One control animal showed a minor irritation reaction to the test material at the 24 hour assessment, the rest of the control animals showed no response. Test animal . All animals showed a minor irritation reaction to the test material at the 24 hour assessment.

Under the conditions of this study, phytic acid was classed as a non-sensitiser.