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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Study was performed in 1993 to the standard methods of the time.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 9 February 1993 to 20 March 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The proposed source chemical (is a mixture of ammonium orthophosphates and ammonium pyrophosphates and is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates and pyrophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate or pyrophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus, the target substance (diammonium dihydrogenpyrophosphate) and the source substance (mixture of ammonium orthophosphates and pyrophosphates) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the toxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection: 17 March 1992 Date of Signature on Certificate: 11 June 1992
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was performed in 1993 to the standard methods of the time. It is not justified to repeat the skin sensitisation endpoint as this study is considered valid for assessment in accordance with REACH and CLP.

Test material

Constituent 1
Reference substance name:
Ammonium polyphosphates impurities not otherwise specified
Molecular formula:
not applicable
IUPAC Name:
Ammonium polyphosphates impurities not otherwise specified
Test material form:
liquid
Details on test material:
The identity of the test material is not reported within the study report itself, however the data is referred to in the Toxicological Risks of Selected Flame Retardant Chemicals (2000), Subcommittee on Flame-Retardant Chemicals, Committee on Toxicology, Board on Environmental Studies and Toxicology, National Research Council. ISBN: 0-309-59232-1. The substance LR-2 is an ‘ammonium polyphosphate’ and the author provides the following additional information with regards to the chemical identity of LR2: ‘Based on information provided by the manufacturer (Stewart Miller, Albright and Wilson, pers. commun., Nov. 1, 1999), a typical species distribution of polyphosphates in LR2 is 20% orthophosphate, 40% pyrophosphate,
Specific details on test material used for the study:
The identity of the test material is not reported within the study report itself, however the data is referred to in the Toxicological Risks of Selected Flame Retardant Chemicals (2000), Subcommittee on Flame-Retardant Chemicals, Committee on Toxicology, Board on Environmental Studies and Toxicology, National Research Council. ISBN: 0-309-59232-1. The substance LR-2 is an ‘ammonium polyphosphate’ and the author provides the following additional information with regards to the chemical identity of LR2: ‘Based on information provided by the manufacturer (Stewart Miller, Albright and Wilson, pers. commun., Nov. 1, 1999), a typical species distribution of polyphosphates in LR2 is 20% orthophosphate, 40% pyrophosphate,

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Supplied by David Hall Limited, Burton-on-Trent, Staffordshire, U.K.
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 331-414 g
- Housing: In groups of up to three in solid-floor polypropylene cages furnished with softwood shavings
- Diet (e.g. ad libitum): Guinea Pig FDl Diet, Special Diet Services limited, Witham, Essex, U.K.) provided ad libitum
- Water (e.g. ad libitum): Mains tap water provided ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 31-56
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hours light

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: intradermal and topical
Vehicle:
other: distilled water; mixture of Freund's complete adjuvant plus distilled water (1:1)
Concentration / amount:
Intradermal induction:
1% (w/v) in distilled water
1% (w/v) in a mixture of Freund's Complete Adjuvant plus distilled water (1: 1)

Topical induction: undiluted as supplied

Topical challenge: 75% and 50% (v/v in distilled water)
Challengeopen allclose all
Route:
other: topical
Vehicle:
other: distilled water; mixture of Freund's complete adjuvant plus distilled water (1:1)
Concentration / amount:
Intradermal induction:
1% (w/v) in distilled water
1% (w/v) in a mixture of Freund's Complete Adjuvant plus distilled water (1: 1)

Topical induction: undiluted as supplied

Topical challenge: 75% and 50% (v/v in distilled water)
No. of animals per dose:
20 animals per dose
Details on study design:
RANGE FINDING TESTS:

a) Selection of concentration for intradermal induction

Two animals were intradermally injected with preparations of test material (10% or 5% w/v in distilled water). The highest concentration that did not cause local necrosis, ulceration or systemic toxicity, was selected for the intradermal induction stage of the main study.

b) Selection of concentration for topical induction

Two guinea pigs (intradermally injected with Freund's Complete Adjuvant nine days earlier) were treated with the undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in distilled water).

The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure, was selected for the topical induction stage of the main study.

c) Selection of concentration for topical challenge

The undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in distilled water) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study ) up to Day 14. The highest non-irritant concentration of the test material and one lower concentration were selected for the
topical challenge stage of the main study .

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Exposure period: 7 days (intradermal), 48 hours (topical)
- Test groups: 1 (20 animals)
- Control group: 1 (10 animals)
- Site: 40 mm x 60 mm on shoulder region of each animal
- Frequency of applications: 3 x intradermal injections at same time, 1 week later same site
- Concentrations:
Intradermal induction:
i) Freund's Complete Adjuvant plus distilled water in the ratio 1: 1.
ii) 1% (w/v) dilution of test material in distilled water
iii) 1% (w/v) dilution of test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water

Topical induction: undiluted as supplied

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 21
- Exposure period: 48 hours
- Test groups: 1 (20 animals)
- Control group: 1 (10 animals)
- Site: An area approximately 50 mm x 70 mm on both flanks of each animal
- Concentrations:75% and 50% (v/v in distilled water)
- Evaluation (hr after challenge): 24 and 48 hours
Challenge controls:
Control group of 10 animals was used
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene

Results and discussion

Positive control results:
The frequency of sensitisation responses was 9 out of 10.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Challenge - 75% v/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Challenge - 75% v/v. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No data.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Challenge - 75% v/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Challenge - 75% v/v. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No data.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Challenge - 50% v/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Challenge - 50% v/v. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No data.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Challenge - 50% v/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No data
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Challenge - 50% v/v. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No data.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Challenge - 75% v/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No data
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Challenge - 75% v/v. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No data.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Challenge - 75% v/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No data
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Challenge - 75% v/v. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No data.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Challenge - 50% v/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No data
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Challenge - 50% v/v. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No data.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Challenge - 50% v/v
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No data
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Challenge - 50% v/v. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No data.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0,25%
No. with + reactions:
9
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.25%
No. with + reactions:
6
Total no. in group:
10

Any other information on results incl. tables

Intradermal and Topical Sighting Tests

A summary of the results of the intradermal sighting test and the individual skin reactions observed in the topical sighting tests are given in Appendices I, II and III.

Based on these results, the following concentrations were selected for the main study:

Intradermal induction: 1% (w/v) in distilled water

Topical induction: undiluted as supplied

Topical challenge: 75% and 50% (v/v) in distilled water

Skin Reactions Observed After Topical Induction

Individual reactions observed at the topical induction sites of test and control animals are given in Appendices IV and V.

Scattered mild redness was elicited by the test material.

Skin Reactions Observed After Topical Challenge

Individual reactions at the challenge sites of test and control animals are given in Tables 1 and 2.

No skin reactions were nated at the challenge sites and vehicle control sites of the test or control group animals at the 24 and 48-hour observations.

Bodyweight

Individual bodyweights and bodyweight gains of test and control animals are given in Appendices VI and VII.

Bodyweight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group in the same period.

Table 1: Individual Skin Reactions in Test Animals at Challenge

Animal Number

Skin Reactions (Hours After Removal of Dressing)

24 hours

48 hours

75%

50%

Vehicle

75%

50%

Vehicle

1

0

0

0

0

0

0

2

0

0

0

0

0

0

3

0

0

0

0

0

0

4

0

0

0

0

0

0

5

0

0

0

0

0

0

6

0

0

0

0

0

0

7

0

0

0

0

0

0

8

0

0

0

0

0

0

9

0

0

0

0

0

0

10

0

0

0

0

0

0

11

0

0

0

0

0

0

12

0

0

0

0

0

0

13

0

0

0

0

0

0

14

0

0

0

0

0

0

15

0

0

0

0

0

0

16

0

0

0

0

0

0

17

0

0

0

0

0

0

18

0

0

0

0

0

0

19

0

0

0

0

0

0

20

0

0

0

0

0

0

Table 2: Individual Skin Reactions in Control Animals at Challenge

Animal Number

Skin Reactions (Hours After Removal of Dressing)

24 hours

48 hours

75%

50%

Vehicle

75%

50%

Vehicle

21

0

0

0

0

0

0

22

0

0

0

0

0

0

23

0

0

0

0

0

0

24

0

0

0

0

0

0

25

0

0

0

0

0

0

26

0

0

0

0

0

0

27

0

0

0

0

0

0

28

0

0

0

0

0

0

29

0

0

0

0

0

0

30

0

0

0

0

0

0

Appendix I: Intradermal sighting test – Summary of Results

Animal Identification

Time of Observation

Concentration of Test Material (% w/v)

Evidence of Local Necrosis

Evidence of Systemic Toxicity

 

A

 

24 hours

 

1

None

None

48 hours

None

None

72 hours

None

None

7 days

None

None

 

B

24 hours

 

5

Necrosis

None

48 hours

Necrosis

None

72 hours

Necrosis

None

7 days

Eschar

None

 

The concentration of the test material selected for the intradermal induction stage of the main study was 1% (w/v) in distilled water.

Appendix II: Topical sighting test for induction application (48-hour exposure) – Individual skin reactions

Animal Identification

Concentration of Test Material (% w/v)

Skin Reactions

(Hours After Removal of Patches)

1

24

48

 

C

 

100

2 Oe

1 Oe

1

75

2

1

0

50

2

1

0

25

1

0

0

 

D

10

2 Oe

1 Oe

1 Oe Ss

75

2

1

0

50

2

1

0

25

1

0

0

 

The undiluted test material was selected for the main study topical induction.

Oe = slight oedema Ss = small superficial scattered scabs

 

Appendex III: Topical sighting test for challenge application (24-hour exposure) – individual skin reactions

Animal Identification

Concentration of Test Material (% w/v)

Skin Reactions

(Hours After Removal of Patches)

1

24

48

 

E

 

100

2 NOe

1 NOe

0 Ss

75

0

0

0

50

0

0

0

25

0

0

0

 

F

10

1 Oe

1 Oe

O Ss

75

0

0

0

50

0

0

0

25

0

0

0

 

The concentrations of the test material selected for the main study topical challenge were 75% and 50% (v/v) in distilled water.

N = area of black-coloured spotted dermal necrosis Ss = small superficial scattered scabs Oe = slight oedema

Appendix IV: Topical induction – Individual Skin Reactions in Test Animals

Animal Number

Skin Reactions (Hours After Removal of Dressing)

1 hour

24 hours

1

1

0

2

1

0

3

1

0

4

1

0

5

1

0

6

1

0

7

1

0

8

1

0

9

1

0

10

1

0

11

1

0

12

1

1

13

1

0

14

1

0

15

1

0

16

1

1

17

1

0

18

1

0

19

1

0

20

1

0

 

Appendix V: Topical Induction – Individual Skin Reactions in Control Animals

Animal Number

Skin Reactions (Hours After Removal of Dressing)

1 hour

24 hours

21

0

0

22

0

0

23

0

0

24

0

0

25

0

0

26

0

0

27

0

0

28

0

0

29

0

0

30

0

0

 

Appendix VI: Individual Bodyweights and Bodyweight Gains of Test Animals

Animal Number

Bodyweight (g)

Bodyweight (g) Increase

Day 0

Day 24

1

366

569

203

2

369

565

196

3

341

535

194

4

408

618

210

5

381

564

183

6

391

551

160

7

346

489

143

8

371

578

207

9

341

571

230

10

380

581

201

11

331

520

189

12

375

566

191

13

382

598

216

14

414

583

169

15

380

594

214

16

333

475

142

17

359

532

173

18

348

500

152

19

401

583

182

20

377

552

175

 

Appendix VII: Individual Bodyweights and Bodyweight Gains of Control Animals

Animal Number

Bodyweight (g)

Bodyweight (g) Increase

Day 0

Day 24

21

363

541

178

22

364

547

183

23

351

545

194

24

347

540

193

25

363

512

149

26

366

517

151

27

356

506

150

28

364

569

205

29

413

646

233

30

383

595

212

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material, Amgard LR2, produced a 0% (0/20) sensitisatio rate and was classified as a non-sensitiser to guinea pig skin.
Executive summary:

A study was performed to assess the skin contact sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 "Skin Sensitisation"- Magnusson and Kligman Maximisation Test referenced as Method B6 in Commission Directive 84/449/EEC (which constitutes Annex V of Council Directive 61/548/EEC).

Twenty test and ten control animals were used for the main study.

Based on the results of the sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

Intradermal induction: 1% (w/v) in distilled water

Topical induction: undiluted as supplied

Topical challenge: 75% and 50% (v/v) in distilled water

The test material produced a 0% (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin.