Phosphoric acid, mono- and di-C6-10-alkyl esters

Endpoint summary

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:

19 September 2011 to 27 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles river (F-69592 L'Arbresle)
- Age at study initiation: 4 weeks old
- Weight at study initiation: 255- 292 g
- Housing: the animals were housed either in groups of 2 or individually in polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted a stainless steel lid with a feeding device and drinking device of 500 mL.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19- 25°C
- Humidity (%): 30- 70%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

PREPARATION OF ANIMALS
The animals were carefully shorn before each test item application:
- On the inter-scapular zone for the induction phase,
- On the dorso-lumbar zone for the challenge phase.
At least 3 hours before the first reading (challenge phase) they were shorn a second time in this dorso-lumbar zone.
The animals were weighed at the beginning and at the end of the study.

Route:

intradermal and epicutaneous

Vehicle:

other: the test item was used freshly prepared in isotonic sodium chloride for the intradermic injections and in distilled water for the topical applications.

Concentration / amount:

Main study:
Induction: Intradermic injection at 0.1% in isotonic soidum chloride and topical application at 50% in distilled water.
Challenge: 0.5% and 0.25% in distilled water

Route:

epicutaneous, occlusive

Vehicle:

other: the test item was used freshly prepared in isotonic sodium chloride for the intradermic injections and in distilled water for the topical applications.

Concentration / amount:

Main study:
Induction: Intradermic injection at 0.1% in isotonic soidum chloride and topical application at 50% in distilled water.
Challenge: 0.5% and 0.25% in distilled water

No. of animals per dose:

Group 1 (negative control): 5
Group 2 (treated): 10

Details on study design:

RANGE FINDING TESTS:
Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC) :
This test was conducted for the purpose of defining a MNNC of the test item which, on intradermal injection during the induction phase, does not risk causing too great a lesion (non-necrotizing concentration), should be well tolerated systemically and should be the highest to cause mild-to-moderate skin irritation.

One animal received on both sides of the spine, a volume of 0.1 mL of the test item, at 4 concentrations: diluted at 25 %, 10 %, 5 % and 2.5 % in isotonic sodium chloride in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after injection.

Due to the necrosis registered, a new animal received on both sides of the spine, a volume of 0.1 mL of the test item, at 4 concentrations: diluted at 1 %, 0.5 %, 0.1 % and 0.05 % in isotonic sodium chloride in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after injection.

The results were confirmed with a new animal. One animal received on both sides of the spine, a volume of 0.1 mL of the test item, at 2 concentrations: diluted at 0.1 % and 0.05 % in isotonic sodium chloride in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after injection.

Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC):
This test, which allowed to evaluate the irritant potential of the test item, defined whether an application of sodium lauryl sulphate would be needed during topical induction phase.
The test item was applied on the dorso-lumbar zone of 1 animal shorn beforehand, with occlusive dressing for 24 h, at 4 different concentrations: diluted at 50 % in distilled water and 25 %, 10 % and 5 % in isotonic sodium chloride.
The results were confirmed with a new animal. The animal received the test item in the same experimental conditions, at 4 different concentrations: diluted at 50 %, 25 %, 10 % and 5 % in distilled water. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after removal of the dressing.

Determination by topical application of the Maximal Non Irritant Concentration (MNIC):
This test was carried out for the purpose of determining the MNIC of the test item without risk of an irritant effect during the challenge phase.
Three animals were treated identically to the animals from Group 1 (negative control) for the induction phase (i.e. isotonic sodium chloride and distilled water).
During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites at concentrations: 4 %, 2 %, 1 % and 0.5 % in distilled water and covered with an occlusive dressing for a period for 24 h.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 h after removal of the occlusive dressing.

MAIN STUDY

Group 1 (negative control): 5 male guinea pigs
Group 2 (treated): 10 male guinea pigs

INDUCTION PHASE:
1st Intradermal Induction:
Day 0: after shearing the scapular zone, 3 pairs of intradermal injections (ID) of 0.1 mL were made on the scapular zone such that an injection from each pair was placed either side of the spine as follows:

GROUP 1 (negative control):
2 ID: Freund’s Complete Adjuvant diluted at 50 % in isotonic sodium chloride;
2 ID: isotonic sodium chloride;
2 ID: a mixture with equal volumes v/v of Freund’s Complete Adjuvant at 50 % and isotonic sodium chloride.

GROUP 2 (treated):
2 ID: Freund’s Complete Adjuvant diluted at 50 % in isotonic sodium chloride;
2 ID: test item at 0.1 % in isotonic sodium chloride;
2 ID: a mixture with equal volumes v/v of Freund’s Complete Adjuvant at 50 % and the test item at 0.2 % in isotonic sodium chloride.

Day 1: Skin reaction evaluation

2nd Topical Induction:
Day 6: the scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulphate at 10 % in thick Vaseline, in order to create a local irritation.
Day 7: a topical application under occlusive dressing for 48 h was performed on the injection sites of each animal. A control of the local irritation was done just before the topical application.
GROUP 1 (negative control): 0.5 mL of distilled water
GROUP 2 (treated): 0.5 mL of the test item at 50 % in distilled water.
Day 9: occlusive dressing removal
Day 10: Skin reaction evaluation
Rest phase: the animals of both groups were left for 10 d.

CHALLENGE PHASE:
Day 20: the experimental procedure of this phase was identical for both groups, GROUP 1 (Negative control) and GROUP 2 (Treated): to the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing for 24 hours of
- 1 sample cup containing the test item diluted at 0.5% (MNIC) in distilled water and to the other side of the spine 1 sample cup containing the test item at 0.25% in distilled water (1/2 MNIC).

Day 21: occlusive dressing removal.

A macroscopic evaluation of the cutaneous reactions (erythema and oedema) was conducted and all the local or systemic reactions are recorded as GROUP 1 (Negative control) and GROUP 2 (Treated) :

Day 22
Approximately 21 hours after removal of the occlusive dressing, the treated zone was shaved, Approximately 3 hours later, the cutaneous reactions were observed and graded according to the scales, given below. (see any other information on materials and methods incl .tables)

Day 23
24 hours later (i.e. 48 hours after removal of the occlusive dressing), a second observation was made.

Challenge controls:

The negative control group was treated identically to the treated group.

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde

Positive control results:

The results of the 3 last positive groups, using alpha-Hexylcinnamaldehyde, carried out as method sensibility are attached (see attached background material). Under the experimental conditions, the reference substance, α-Hexylcinnamaldehyde is classified as a skin sensitiser.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.5 %

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

slight erythema in 20 % (1/5) of the animals.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0.5 %. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: slight erythema in 20 % (1/5) of the animals..

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.5 %

No. with + reactions:

0

Clinical observations:

No reaction was noted.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.5 %. No with. + reactions: 0.0. Clinical observations: No reaction was noted..

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.5 %

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

slight erythema in 10 % (1/10) of the animals from the treated group on the treated area.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.5 %. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: slight erythema in 10 % (1/10) of the animals from the treated group on the treated area..

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.5 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No reaction was noted.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.5 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No reaction was noted..

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.25 %

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

slight erythema in 20 % (1/5) of the animals from the negative group on the treated area.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0.25 %. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: slight erythema in 20 % (1/5) of the animals from the negative group on the treated area. .

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.25 %

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No reaction was noted.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.25 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No reaction was noted..

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.25 %

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

slight erythema in 10 % (1/10) of the animals from the treated group on the treated area.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.25 %. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: slight erythema in 10 % (1/10) of the animals from the treated group on the treated area. .

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.25 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No reaction was noted.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.25 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No reaction was noted..

Results

Concentrations selected

Preliminary studies

MNNC determination:

The results are given in Table 1 (see attached background material).

No necrosis has been observed for the 2 animals, at the concentration of 0.1 %. Based on this a concentration of 0.1% was selected for intradermal induction in the main study of the Group 2 treated animals.

Pre-MNIC determination:

The results are given in Table 2 (see attached background material).

24 h after the removal of the occlusive dressings, slight to well defined erythema was recorded on the treated area at 50 %, 25 % and 10 % in 2 animals and slight erythema was recorded on the treated area at 5 % in 1 animal.

Based on these results, the concnetration selected was 50% for the topical induction of the Group 2 with sodium lauryl sulphate pre treatment (10% in vaseline) and the MNIC deternmination began at the concentration 4%.

MNIC determination:

The results are given in Table 3 (see attached background material).

Slight to well defined erythema associated or not with a very slight to slight oedema was recorded on the treated area at 4%, 2% and 1%, 24 and 48h after patch removal. No cutaneous reaction was recorded on the treated areas at 0.5%

Based on these results, the concentrations selected for the challenge phase of the main study were 0.5% (MNIC) and 0.25% (1/2 MNIC)

Main Study

Induction phase Group 2: the induction phase was performed by intradermal injection on D0 with the test item at 0.1 % in isotonic sodium chloride and by topical application on D7 with the test item at 50 % in distilled water after treatment with sodium lauryl sulphate 24 h earlier. No cutaneous reactions was recorded after the 1^stinduction phase. During the 2^ndinduction, dryness was noted in 7 animals (7/10) and a scab was noted in 3 animals (3/10), 24 h after the removal of the occlusive dressing.

Induction phase Group 1: the induction phase was performed by intradermal injection on D0 with isotonic sodium chloride and by topical application on D7 with distilled water after treatment with sodium lauryl sulphate 24 h earlier. No cutaneous reaction was recorded after the induction phases.

Challenge phase Groups 1 & 2: the test item was diluted at 0.5 % and 0.25 % in distilled water.

Sensitising potential assessment

Overall results of the challenge phase with the test item (readings at 24 and 48 hours) are given in table 4 (see attached backgound material).

Individual scores of macroscopic evaluations performed during challenge phase with the test item are given in table 5 (see attached background material).

Slight erythema was observed on the area treated with the test item at either 0.5% or 0.25% in 10% (1/10) of Group 2 (treated) animals 24 hours after the challenge phase. No reaction was noted in Group 2 (treated) animals treated with test item at either 0.5% or 0.25% at the reading time 48 hours.

Slight erythema was observed in 10% (1/10) of the animals from the treated group, 24 hours after the challenge phase, on the treated area with the test item at 0.25%. No reaction was noted at the reading time 48 hours.

Slight erythema was observed in 20% (1/5) of the animals from the negative control group, 24 hours after the challenge phase, on the treated area with the test item at 0.25%. No reaction was noted at the reading time 48 hours.

Body Weights

The body weight of negative control animals (Group 1) and treated animals (Group 2) is presented in tables 6 and 7 respectively (see attached background material).

No abnormality was recorded in the body weight gain of either groups.

Mortality

No mortality was registered during the main study.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the substance was found not to be a skin sensitiser.

Executive summary:

The aim of the study was to evaluate the possible cutaneous allergenic activity of the test item after intradermal and topical administration in guinea pigs. The experimental protocol was established according the OECD guideline No. 406 dated July 17th, 1992 and the test method B.6 of the council regulation No. 440/2008.

Induction of 10 Guinea Pigs in the treated group (Group 2) with the test item Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) was by intradermal injections with 0.1% test item in isotonic sodium chloride and in Freund’s Complete Adjuvant followed one week later by treatment with sodium lauryl sulphate and 24 hours thereafter topical application at 50%

test item in distilled water.

A negative control group (5 guinea pigs) received intradermal injections of isotonic sodium chloride and Freund’s Complete Adjuvant in isotonic sodium chloride followed one week later by treatment with sodium lauryl sulfate and 24 hours thereafter topical application of distilled water.

Slight erythema was observed on the area treated with the test item at either 0.5% or 0.25% in 10% (1/10) of Group 2 (treated) animals 24 hours after the challenge phase. No reaction was noted in Group 2 (treated) animals treated with test item at either 0.5% or 0.25% at the reading time 48 hours.

Slight erythema was observed on the area treated with the test item at either 0.5% or 0.25% in 20% (1/5) of Group 1 (negative control) animals 24 hours after the challenge phase. No reaction was noted in Group 1 (negative control) animals treated with test item at either 0.5% or 0.25% at the reading time 48 hours.

In conclusion, in view of these results, under these experimental conditions, the test item is not a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The aim of the study was to evaluate the possible cutaneous allergenic activity of the test item after intradermal and topical administration in guinea pigs. The experimental protocol was established according the OECD guideline No. 406 dated July 17th, 1992 and the test method B.6 of the council regulation No. 440/2008.

Induction of 10 Guinea Pigs in the treated group (Group 2) with the test item Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) was by intradermal injections with 0.1% test item in isotonic sodium chloride and in Freund’s Complete Adjuvant followed one week later by treatment with sodium lauryl sulphate and 24 hours thereafter topical application at 50% test item in distilled water.

A negative control group (5 guinea pigs) received intradermal injections of isotonic sodium chloride and Freund’s Complete Adjuvant in isotonic sodium chloride followed one week later by treatment with sodium lauryl sulfate and 24 hours thereafter topical application of distilled water.

Slight erythema was observed on the area treated with the test item at either 0.5% or 0.25% in 10% (1/10) of Group 2 (treated) animals 24 hours after the challenge phase. No reaction was noted in Group 2 (treated) animals treated with test item at either 0.5% or 0.25% at the reading time 48 hours.

Slight erythema was observed on the area treated with the test item at either 0.5% or 0.25% in 20% (1/5) of Group 1 (negative control) animals 24 hours after the challenge phase. No reaction was noted in Group 1 (negative control) animals treated with test item at either 0.5% or 0.25% at the reading time 48 hours.

In conclusion, in view of these results, under these experimental conditions, the test item is not a skin sensitiser.

Migrated from Short description of key information:
The sensitising potential of the substance was assessed according to OECD Guideline 406. Under the conditions of this study the substance was found not to be a skin sensitiser.

Justification for selection of skin sensitisation endpoint:
The study has been conducted according to OECD Guideline 406 and GLP and is adequately reported. The study has been assigned a reliability 1.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In view of the results from an OECD 406 study, under these experimental conditions, the test item is not a skin sensitiser.

In accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures, the test item must not be classified. No hazard statement or signal word is required.