Registration Dossier

Administrative data

Description of key information

Skin sensitisation: not sensitising (OECD 406; GLP)

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:
2015-12-14 to 2016-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992-07-17
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The data requirements as laid down in the REACH regulation Annex VII, foresee “the murine local lymph node assay (LLNA) as the first choice method for in vivo testing. Only in exceptional circumstances should another test be used. Justification for the use of another in vivo test (e.g. Guinea pig maximisation test (GPMT)) shall be provided.”

According to the OECD guideline 429 (2010), limitation for not using the LLNA test is the possibility of false positive results due to skin irritation. If a substance has skin irritation properties another in vivo test such as the GPMT test according to OECD guideline 406 (1992) should be considered.

Although the in vivo skin irritation testing in rabbits (according to OECD 404 and GLP) did not reveal irritating properties, a severe conjunctival reaction with a Draize score of up to 3 was received in the in vivo eye irritation testing. Since the skin physiology of the rabbit skin and the conjunctival membrane of the rabbit eye is equivalent, but with the important difference that the conjunctival membrane is lacking the stratum corneum. The stratum corneum consists of several layers of dead corneocytes, which function as a barrier to protect underlying tissue from chemical stress. Since a significant positive response was received for the conjunctivae in the eye irritation assay, one has to assume that the test substance has the intrinsic irritating property for biological membranes, which does not occur in skin with intact protective layer.

It was therefore decided not to perform the in vivo LLNA assay, since irritating properties would have led to a false positive test results. In order to avoid the possibility of a false positive result in the LLNA test, it was decided to conduct the GPMT test (OECD 406, 1992) with triiron bis(orthophosphate) instead of the LLNA.
Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Crl:(HA)BR
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 4 - 6 weeks old
- Weight at study initiation: 336.1 - 416.7 g
- Housing: housed in groups of up to ten
- Diet (ad libitum): commercial feeding mixture (Mühle Knull, Rostock, Germany)
- Water (ad libitum): tap water (drinking quality, supplemented with 1 g/L vitamin C)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Relative humidity: 30 - 70 %
- Air changes: 16/hours
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
other: distilled water
Concentration / amount:
1 %
Day(s)/duration:
day 0
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
100 %
Day(s)/duration:
day 7 (exposure duration: 48 hours)
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
100 %
Day(s)/duration:
day 22 (exposure duration: 24 hours)
No. of animals per dose:
10 female guinea pigs
Details on study design:
The vehicle, distilled water, was selected based on preliminary tests. The test material was not soluble in distilled water and injected as suspension. For topical application the test material ws moistened with distilled water. The preparations of the test material were made immediately prior to each application procedure.

RANGE FINDING TESTS:
The irritation response to intradermal injection of various concentrations of the test substance was examined in three guinea pigs. An area of the flanks was clipped free from hair with electric clippers. Amount of 0.1 mL of the selected test concentrations (5 %; 2.5 %; 1 % and 0.5 % suspensions of the test material in distilled water) were applied by intradermal injection. 24 and 48 hours after injection the animals were examined for signs of irritation according to the Magnusson and Kligman grading scale.
The irritation response to topical treatment of various concentrations of the test substance was examined in three further guinea pigs. The flanks of the animals were clipped. Filter paper fully-loaded with the test substance (100 %; 50 %; 25 % in distilled water) was attached to the skin of the guinea pigs and held in contact by an occlusive dressing for 24 hours. The animals were observed and examined for sings of irritation according to the Magnusson and Kligman grading scale approx. 24 hours and 48 hours after removing the test material.

Resullts:
- intradermal induction: the concentration of 1 % of the test material in distilled water (suspension) was systemically well-tolerated and caused a mild - moderate skin irritation, this concentration was used for the main test.
- topical application (induction): the concentration of 100 % of the test material moistened with distilled water was systemically well-tolerated and did not cuase a skin irritation, this concentration was used for the main test.
- topical application (challenge): a concentration of 100 % of the test material moistened with distilled water was used. This concentration was well-tolerated systemically and did not cause skin irritation.

Please also refer for information on tested doses to the field "Any other information on materials and methods incl. tables" below.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal injection and topical application)
- Site: area of dorsal skin from the scapular region (approx. 4 cm x 6 cm) was clipped free of hair
- Frequency of applications: three pairs of intradermal injections of 0.1 mL volume were given to the animals. Seven days after intradermal induction the test substance was topically applied using filter paper. Because the agent was non-irritating at this concentration, the region was pretreated with 0.5 mL 10 % sodium lauryl sulphate in vaseline for 24 hours. The control animals were treated with vehicle similarly.
- Exposure period: 48 hours (topical application)
- Concentrations:
Intradermal:
i) 0.1 mL 1:1 mixture (v/v) FCA/water
ii) 0.1 mL test substance suspension of 1 % in distilled water
iii) 0.1 mL test substance at the selected concentration formulated in a 1:1 mixture (v/v) FCA/water
The control animals were treated with the vehicle similarly.

Topical application:
100 % finely pulverised test substance moistened with distilled water
The control animals were treated with the vehicle similarly.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (topical application three weeks after the intradermal induction)
- Exposure period: 24 hours
- Site: flank were cleared of hair of test and control animals
- Concentrations: 100 % finely pulverised test substance moistened with distilled water
- Evaluation (hr after challenge): 24 and 48 hours after removal of the patch
Evaluation according to the Magnusson and Kligman grading scale.

EXAMINATIONS:
- clinical observations: clinical symptoms were recorded over the period of observations
- body weight: at the start and at the end of the test
Challenge controls:
5 guinea pigs were used as control animals
Challenge dose: 100 % finely pulverised test substance moistened with distilled water
Positive control substance(s):
yes
Remarks:
hexylcinnamaldehyde (5 % for intradermal induction, 75 % for topical induction and 55 % for challenge)(study conduct: 2015-09-23 to 2015-10-16)
Positive control results:
90 % of the animals treated with the positive control (55 % hexylcinnamalde hyde in vaseline) showed a skin reaction with numerical grading from 0-1 up to 2 according to Magnusson and Kligman grading scale.
The positive control showed a sensitisation effect and the validity of the test system.
Key result
Reading:
1st reading
Group:
test chemical
Dose level:
100 % of the test item moistened with distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
The animals did not show any visible clinical symptoms over the period of observation. All animals showed the expected body weight development.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 % of the test item moistened with distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
The animals did not show any visible clinical symptoms over the period of observation. All animals showed the expected body weight development.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 % of the test item moistened with distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
The animals did not show any visible clinical symptoms over the period of observation. All animals showed the expected body weight development.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 % of the test item moistened with distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
The animals did not show any visible clinical symptoms over the period of observation. All animals showed the expected body weight development.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
55 % hexylcinnamalde hyde in vaseline
No. with + reactions:
8
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
55 % hexylcinnamalde hyde in vaseline
No. with + reactions:
9
Total no. in group:
10
Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not a skin sensitiser.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation

The substance does not possess a skin sensitisation potential based on an absence of adverse effects in a reliable guinea pig maximization test (OECD 406).

For justification for type of information please refer to attachements.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Respiratory sensitisation

In a weight of evidence approach, all available information was evaluated with the outcome that triiron orthophosphate does not possess a respiratory sensitising potential. The substance does not possess a skin sensitisation potential based on an absence of adverse effects in a reliable guinea pig maximization test (OECD 406). No effects for respiratory sensitisation were seen in an acute inhalation toxicity study with 14 -day post exposure observation in rats. In the industrial production process and during handling of the registered substance, no cases of respiratory hypersensitivity have been observed. Classification as respiratory sensitiser does not appear justified.

Justification for classification or non-classification

Skin sensitisation

The substance does not possess a skin sensitisation potential based on an absence of adverse effects in a reliable guinea pig maximization test (OECD 406).

Respiratory sensitisation

In a weight of evidence approach, all available information was evaluated with the outcome that triiron orthophosphate does not possess a respiratory sensitising potential. The substance does not possess a skin sensitisation potential based on an absence of adverse effects in a reliable guinea pig maximization test (OECD 406). No effects for respiratory sensitisation were seen in an acute inhalation toxicity study with 14 -day post exposure observation in rats. In the industrial production process and during handling of the registered substance, no cases of respiratory hypersensitivity have been observed.Classification as respiratory sensitiser does not appear justified.