[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

Endpoint summary

Description of key information

In the key skin sensitisation study for the Category member ATMP-xNH4, conducted according to OECD Test Guideline 406 and in compliance with GLP, ATMP-xNH4 was not sensitising to the skin of guinea pigs (SafePharm Laboratories, 1995).

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:

27.03.1995 to 05.05.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An LLNA study was not performed because there is an existing reliable study for skin sensitisation using the Guinea Pig Maximisation test method.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: David Hall Limited, Staffordshire, UK.
- Age at study initiation:8-12 weeks
- Weight at study initiation: 334-430 g
- Housing: Singly or in pairs in solid-floor polypropylene cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Minimum five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-28
- Humidity (%): 51-68
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 27.03.1995 To 05.05.1995

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

Intradermal induction: 10% w/v in distilled water; 10% w/v in a mixture of FCA plus distilled water (1:1).
Topical induction: undiluted.
Topical challenge: 100% and 75% v/v in distilled water.

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

Intradermal induction: 10% w/v in distilled water; 10% w/v in a mixture of FCA plus distilled water (1:1).
Topical induction: undiluted.
Topical challenge: 100% and 75% v/v in distilled water.

No. of animals per dose:

20 test and 10 control used for the main study.

Details on study design:

RANGE FINDING TESTS: Four concentrations of test substance were investigated (1, 5, 10 and 25% w/v in distilled water). A total of four guinea pigs were used, each animal received four 0.1 ml injections of only one concentration of the test substance. The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and seven days after the injection according to the Draize scale. Oedema was not assessed. Evidence of systemic toxicity was recorded. The highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, was selected for the intradermal induction stage of the main study.
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant 15 days earlier) were treated with the undiluted test substance and three preparations of the test substance (75, 50 and 25%) v/v in distilled water). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours.The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.
The undiluted test substance and three preparations of the test substance (75, 50 and 25% v/v in distilled water) were applied to the clipped flanks of two guinea pigs under occlusive dressings for 24 hours. The animals did not form part of the main study, but had been treated identically to the controls of the main study up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest non-irritant concentration of test substance and one lower concentration were selected for the topical challenge stage of the main study.
MAIN STUDY
A. INDUCTION EXPOSURE
Shortly before treatment on Day 0 the hair was removed from an area on the shoulder region of each animal. A row of three injections (0.1ml) each was made on each side of the mid-line. The injections were: a) FCA plus distilled water (1:1); b) a 10% w/v formulation of the test substance in distilled water; c) a 10% w/v formulation of the test substance in a 1:1 preparation of FCA plus distilled water. Approximately 24 and 48 hours after the intradermal injection, the degree of erythema at the injection sites was evaluated. One week later (Day 7), the same area on the shoulder region was clipped again and treated with a topical application of the undiluted test substance (under an occlusive dressing for 48 hours). The degree of erythema and oedema was quantified at one and 24 hours following removal of the patches.
Induction of the control animals used an identical procedure as the test animals, except the injections were: a) FCA plus distilled water in the ratio 1:1; b) distilled water; c) 50% w/v formulation of distilled water in a 1:1 mixture of FCA/distilled water. The topical applications followed the same procedure as for the test animals, but nothing was applied to the patch. Skin reactions were quantified.

B. CHALLENGE EXPOSURE
Shortly before treatment on Day 21, an area of skin on both flanks of each animal was clipped free of hair. A square filter paper patch saturated with the undiluted test substance was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test substance at a concentration of 75% v/v in distilled water was similarly applied to a skin site on the left shorn flank. The patches were covered with an occlusive dressing. After 24 hours the dressing was removed. The challenge sites were swabbed with cotton wool soaked in distilled water to remove residual material. Prior to the observation period, the flanks were clipped to remove regrown hair.
Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified, and any other reactions recorded. The percentage of test animals that showed a more severe reaction at the test substance challenge site than the most severe reaction observed in the control animals, was compared using the scale: 0% = non-sensitiser; >0-8% = weak sensitiser; >8-28 = mild sensitiser; >28-64% = moderate sensitiser; >64-80% = strong sensitiser; >80-100% = extreme sensitiser.

Challenge controls:

Negative controls only.

Positive control substance(s):

no

Positive control results:

No positive control.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75 and 100%

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 chemical

Dose level:

75 and 100%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

distilled water

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

distilled water

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

positive control

Dose level:

N/a

No. with + reactions:

0

Total no. in group:

0

Remarks on result:

not measured/tested

Body weights of the guinea pigs in the test group between Day 0 and 24, were comparable to those observed in the control groups over the same period.

Interpretation of results:

GHS criteria not met

Conclusions:

In the skin sensitisation study, conducted according to OECD Test Guideline 406 and in compliance with GLP, ATMP-xNH4 was not sensitising to the skin of guinea pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the key skin sensitisation study for the Category member ATMP-xNH4, conducted according to OECD Test Guideline 406 and in compliance with GLP, ATMP-xNH4 was not sensitising to the skin of guinea pigs (SafePharm Laboratories, 1995).

At induction, shortly before treatment on Day 0 the hair was removed from an area on the shoulder region of each animal. A row of three injections (0.1 ml) each was made on each side of the mid-line. The injections were: a) FCA plus distilled water (1:1); b) a 10% w/v formulation of the test substance in distilled water; c) a 10% w/v formulation of the test substance in a 1:1 preparation of FCA plus distilled water. Approximately 24 and 48 hours after the intradermal injection, the degree of erythema at the injection sites was evaluated. One week later (Day 7), the same area on the shoulder region was clipped again and treated with a topical application of the undiluted test substance under an occlusive dressing for 48 hours. The degree of erythema and oedema was quantified at one and 24 hours following removal of the patches.

Induction of the control animals used an identical procedure as the test animals, except the injections were: a) FCA plus distilled water in the ratio 1:1; b) distilled water; c) 50% w/v formulation of distilled water in a 1:1 mixture of FCA/distilled water. The topical applications followed the same procedure as for the test animals, but nothing was applied to the patch. Skin reactions were quantified.

At challenge, shortly before treatment on Day 21, an area of skin on both flanks of each animal was clipped free of hair. A square filter paper patch saturated with the undiluted test substance was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test substance at a concentration of 75% v/v in distilled water was similarly applied to a skin site on the left shorn flank. The patches were covered with an occlusive dressing. After 24 hours the dressing was removed. The challenge sites were swabbed with cotton wool soaked in distilled water to remove residual material. Prior to the observation period, the flanks were clipped to remove regrown hair.

Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified, and any other reactions recorded. The percentage of test animals that showed a more severe reaction at the test substance challenge site than the most severe reaction observed in the control animals, was compared using the scale: 0% = non-sensitiser; >0-8% = weak sensitiser; >8-28 = mild sensitiser; >28-64% = moderate sensitiser; >64-80% = strong sensitiser; >80-100% = extreme sensitiser (SafePharm Laboratories, 1995).

In a supporting skin sensitisation study, conducted according to a protocol similar to OECD Test Guideline 406 but not in compliance with GLP, ATMP-H (without information on active acid content) was not sensitising to the skin of guinea-pigs (Henkel, 1984).

At intradermal induction phase, 3 injections to the skin occurred for of 20 female guinea-pigs and another 20 guinea-pigs which served as a control group. For the test group, the first injection contained 0.1 mL of Freund’s adjuvant, the second injection contained 0.1 mL of the test material (as an aqueous solution) and the third injection contained 0.1 mL of a mixture of Freund’s adjuvant and test material as a 1:1 ratio with a final concentration of 5%.

The control animals also received 3 injections, the first injection contained 0.1 mL of Freund’s adjuvant, the second injection contained 0.1 mL of an aqueous solution and the third injection contained 0.1 mL of a mixture of Freund’s adjuvant and the aqueous solution in a 1:1 ratio. A week after the initial administration and during the epicutaneous induction phase the test material was applied epicutaneously further down the torso at 5% concentration in Vaseline. After 48 hours, the dressings were removed. The control animals were treated with Vaseline only. 14 days after the induction treatment, challenge was performed on a on the shaved flanks of each animal. Application of 0.1 mL of the 5% test substance in aqueous solution occurred to the right flank and 0.1 mL of the aqueous solution only was applied to the left flank under a patch for 24 hours.

During the treatment free period, the Freund’s adjuvant resulted in necrosis and later scar tissue. After the epicutaneous induction, general redness was observed and the injection sites were blood stained. Crust formation was present at injection sites in both the test and control animals. Moreover, one animal in the control group died. No evidence of sensitisation was observed after challenge. After removal of the patch, one animal had white dots in the left eye (Henkel, 1984).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information there is no requirement for the classification and labelling of [nitrilotris(methylene)trisphosphonic acid], ammonium salt in accordance with the current EU guideline.