Trimanganese bis(orthophosphate)

Endpoint summary

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:

08 July - 15 August 2014

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.

The poor solubility (see water solubility) and the granulometry of the test item manganese phosphate, topical application to the mouse ear was difficult to achieve. Due to the difficult feasibility of the LLNA assay, it was decided to perform the GPMT assay, which allows to assess the skin sensitising properties of difficult to test substances.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature 15 - 25 °C

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 300.7-358.3 g
- Housing: test animals were housed in groups of up to ten.
- Diet: commercial feeding mixture (Mühle Knull, Rostock, Germany), ad libitum
- Water: tap water (drinking quality, supplemented with 1 g/L vitamin C), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.5 % of the test item

Day(s)/duration:

day 0

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

100% of the test item

Day(s)/duration:

Day 7 (duration 48 hours)

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

100 % of the test item

Day(s)/duration:

Day 21 (duration: 24 hours)

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

5 (negative control)
10 (test group)

Details on study design:

RANGE FINDING TESTS:
The appropriate test material concentrations for intradermal and epicutaneous induction and epicutaneous challenge exposures were determined in a preliminary test using 6 additional FCA-treated animals.
For intradermal exposure, animals were given the test material at 5, 2.5, 1 and 0.5% suspensions in water by intradermal injections (0.1 mL). Animals were examined for signs of skin irritation at 24 and 48 h post-injection according to the Magnusson and Kligman grading scale.
For topical exposure, animals were treated with the test material at 100, 50 and 25% in distilled water for 24 h under occlusive conditions. Irritation responses were assessed at 24 and 48 h after patch and substance removal.
Based on the results of the preliminary test (see Table 1), in the main test, 0.5% test material in water and 100% test material moistened with water were selected for intradermal and topical treatment, respectively.
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections 0.1 mL)
Injection 1: 1:1 mixture (v/v) FCA/water
Injection 2: 0.5% test substance in water
Injection 3: 0.5% test substance in a 1:1 mixture (v/v) FCA/water
- Negative control group:
Intradermal (3 pairs of injections, each 0.1 mL)
Injection 1: 1:1 mixture (v/v) FCA/water
Injection 2: water
Injection 3: 1:1 mixture (v/v) FCA/water

Epicutaneous:
Test group: test substance at 100%
Negative control: water

- Site: scapular region (intradermal + epicutaneous)
- Frequency of applications: single
- Duration: Days 0-8 (on Day 6, one day prior to epicutaneous induction, the shorn skin of all animals in each group was treated with 0.5 mL of 10% sodium lauryl sulphate vaseline, in order to create a local irritation).

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day(s) of challenge: 21 (challenge)
- Exposure period: 24 h
- Test groups: 100% test substance moistened with water
- Control group: 100% test substance moistened with water
- Site: flank region
- Concentrations: 100%
- Evaluation (hr after challenge): 48 and 72 h

Positive control substance(s):

yes

Remarks:

hexyl cinnamic acid (CAS No 101-86-0, routinely evaluated every 6 month at challenge concentrations of 55% in vaseline)

Positive control results:

Hexyl cinnamic acid (at challenge concentration of 55% in vaseline) induced skin sensitisation reactions in 90% of the treated animals.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% of the test item

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were noted. All animals showed the expected body weight development. The animals did not show any visible clinical symptoms over the period of observation.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% of the test item

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were noted. All animals showed the expected body weight development. The animals did not show any visible clinical symptoms over the period of observation.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% of the test item

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were noted. All animals showed the expected body weight development. The animals did not show any visible clinical symptoms over the period of observation.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% of the test item

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were noted. All animals showed the expected body weight development. The animals did not show any visible clinical symptoms over the period of observation.

Animal weights

Table 3: Individual animal weights (g) at start / test end (test group)

Animal	Test start	Test end	Body weight change
1	315.0	389.5	74.5
2	320.6	401.5	80.9
3	306.3	381.9	75.6
4	304.6	378.0	73.4
5	346.6	432.8	86.2
6	300.8	392.5	91.7
7	323.9	404.2	80.3
8	301.5	361.5	60.0
9	301.8	362.9	61.1
10	314.3	389.4	75.1

 

Individual weight of control group

Table 4: Individual animal weights (g) at test start and at test end (control group)

Animal	Test start	Test end	Body weight change
K1	300.7	376.2	75.5
K2	309.3	381.7	72.4
K3	358.3	420.8	62.6
K4	328.2	405.9	77.7
K5	339.4	418.4	79.0

 

Table 5: Skin reactions of test animals after treatment with the test material

Animal	Numerical grading after
	24 h	48 h
1	0	0
2	0	0
3	0	0
4	0	0
5	0	0
6	0	0
7	0	0
8	0	0
9	0	0
10	0	0

 

Table 6: Skin reactions of control animals after treatment with the test material

Animal	Numerical grading after
	24h	48h
K1	0	0
K2	0	0
K3	0	0
K4	0	0
K5	0	0

 

Table 7: Skin reactions of animals after challenge treatment with HCA 55 % in vaseline

Animal	Numerical grading after
	24 h	48 h
1	1	1
2	0	0
3	1-2	1-2
4	1	1
5	1-2	2
6	1	1
7	1-2	1-2
8	0-1	1
9	1-2	1
10	1	1

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:

In accordance with Annex VII, Section 8.3, Column 2 of Regulation (EC) No 1907/2006 (REACH), the Murine Local Lymph Node Assay (LLNA) is the first-choice method for in vivo testing of skin sensitisation. Only in exceptional circumstances should another test be used. Justification for the use of another test shall be provided.

Paragraphs 4 and 5 of OECD Guideline 429 (as adopted on 22 July 2010) read as follows:

“4. The LLNA provides an alternative method for identifying potential skin sensitizing test substances. This does not necessarily imply that in all instances the LLNA should be used in place of guinea pig tests (i.e. TG 406) […], but rather that the assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation. The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the identity and chemical structure of the test substance; its physicochemical properties; the results of any other in vitro or in vivo toxicity tests on the test substance; and toxicological data on structurally related test substances. This information should be considered in order to determine whether the LLNA is appropriate for the test substance (given the incompatibility of limited types of test substances with the LLNA- see paragraph 5) and to aid in dose selection.

5. The LLNA is an in vivo method and, as a consequence, will not eliminate the use of animals in the assessment of allergic contact sensitizing activity. It has, however, the potential to reduce the number of animals required for this purpose. Moreover, the LLNA offers a substantial refinement (less pain and distress) of the way in which animals are used for allergic contact sensitization testing. The LLNA is based upon consideration of immunological events stimulated by chemicals during the induction phase of sensitization. Unlike guinea pig tests (i.e. TG 406) […] the LLNA does not require that challenge-induced dermal hypersensitivity reactions be elicited. Furthermore, the LLNA does not require the use of an adjuvant, as is the case for the guinea pig maximisation test […]. Thus, the LLNA reduces animal pain and distress. Despite the advantages of the LLNA over TG 406, it should be recognised that there are certain limitations that may necessitate the use of TG 406 […] (e.g. false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants [such as some surfactant type chemicals] […], or solubility of the test substance). In addition, test substance classes or substances containing functional groups shown to act as potential confounders […] may necessitate the use of guinea pig tests (i.e. TG 406) […]. Further, based on the limited validation database, which consisted primarily of pesticide formulations, the LLNA is more likely than the guinea pig test to yield a positive result for these types of test substances […]. However, when testing formulations, one could consider including similar test substances with known results as benchmark test substances to demonstrate that the LLNA is functioning properly […]. Other than such identified limitations, the LLNA should be applicable for testing any test substances unless there are properties associated with these test substances that may interfere with the accuracy of the LLNA.”

Trimanganese bis(orthophosphate) is an inorganic salt of the metal manganese and orthophosphate. The water solubility of trimanganese bis(orthophosphate) is low (13.8 mg/L). Dermal absorption is therefore anticipated to be low (ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7c: Endpoint specific guidance. Version 2.0, November 2014). Based on the identity/chemical structure and physicochemical properties, testing for skin sensitisation by means of a Local Lymph Node Assay (OECD 429) is considered to be inappropriate, as it may underestimate the skin sensitising potential of the test substance, leading to a false negative result, due to a low dermal absorption and hence low exposure. For this reason, the Guinea Pig Maximization Test, which involves intradermal injection of the test substance for induction thus ensuring exposure beneath the skin surface, is considered to be the most appropriate method for assessing the skin sensitising potential of this particular substance.

The skin sensitising potential of trimanganese bis(orthophosphate) was evaluated in a Guinea Pig Maximization Test (GPMT) conducted in accordance with OECD Guideline 406 and GLP (Grümmer, 2014). Preliminary tests were conducted to determine the test substance concentrations for intradermal and epicutaneous applications in the main study. Test animals (10 female Dunkin Hartley guinea pigs) were intradermally induced with the test substance as a 0.5% suspension in water (Day 0), and topically induced with the test substance at 100% moistened with water (Day 7) for 48 h. Control animals (5 females) were treated similarly with distilled water. Control and test animals were challenged by topical application of the test substance at 100% moistened in water (Day 21) for 24 h. For topical induction and challenge applications, the skin of the test animals was pre-treated with 10% sodium lauryl sulphate in vaseline for 24 h, respectively. Skin reactions were examined and evaluated 24 and 48 h after challenge patch removal (i.e. 48 and 72 h after challenge application). No skin reactions were noted at the challenge sites of the control and test animals at the observation time points. Hence, the test material did not induce any skin reactions in intradermally and topically induced guinea pigs after challenge treatment.

Therefore, the test material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not skin sensitising.

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 trimanganese bis(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 trimanganese bis(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.