Phosphonic acid, monoethyl ester, sodium salt (1:1)

Endpoint summary

Description of key information

Guinea pig maximization test (OECD 406); GLP; Intradermal induction 1%, Challenge 25%; not skin sensitizing

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:

17 May - 21 Jun 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

(adopted in July 1992)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

(adopted in July 1996)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(adopted in August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Republique Francaise, Premier Ministre, Groupe Interministeriel Des Produits Chimiques, Paris Cedex, France

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was initiated prior to the implementation of the LLNA method.

Species:

guinea pig

Strain:

other: Hartley Crl: (HA) BR

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, L'Arbresle, France
- Age at study initiation: 1 - 2 months
- Weight at study initiation: 352 ± 27 g (males), 354 ± 20 g (females)
- Housing: individually in polycarbonate cages with stainless steel lid and autoclaved sawdust
- Diet: 106 pelleted diet (UAR, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: filtered water (FG Millipore membrane (0.22 micron) , ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 17 May To: 21 June 2002

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

1%

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

40%

Day(s)/duration:

48 h

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25%

Day(s)/duration:

On day 22 for 24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

preliminary test: 2 (per sex, test group)
main study: 10 (per sex, test group), 5 (per sex, control)

Details on study design:

RANGE FINDING TESTS:
Intradermal:
24 hours before treatment, the dorsal region of the animals was clipped. Injections were performed in the interscapular region.
Test concentrations: 0.1, 1, 5, 10 and 25% (w/w)
Evaluation of cutaneous reactions: 24 and 48 h, 6 days after the injections

Epicutaneous:
24 hours before treatment, both flank regions of the animals were clipped and shaved. The filter paper of a chamber (Finn Chamber®) was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours.
Test concentrations: 25 and 40% (w/w, 40% (maximal practicable concentration))
Evaluation of cutaneous reactions: 24 and 48 h after treatment or removal of the dressings

Criteria for the selection of concentrations suitable for the main test
- the concentrations should be well-tolerated (systemically and locally)
- intradermal injections should cause moderate irritant effects (no necrosis or ulceration of the skin)
- cutaneous application for the induction should cause a weak to moderate skin reaction or be the maximal practicable concentration
- cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effects.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline
Injection 2: test substance in physiological saline (1%, w/w)
Injection 3: test substance (1%, w/w) in a 1:1 mixture (v/v) FCA/physiological saline
Epicutaneous: 40% (w/w)
- Control group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline
Injection 2: physiological saline
Injection 3: physiological saline (50%, w/v) in a 1:1 mixture FCA/physiological saline
Epicutaneous: physiological saline
- Site: interscapular region (clipped on Day -1 and 7; clipped and shaved on Day 21 (flanks))
- Frequency of applications: every 7 days
- Duration: Days 1 - 10 (including maintainance of the occlusive dressing for 48 h)
- Concentration: 1% (intradermal), 40% (epicutaneous)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 h
- Test groups: test substance (right flank) and vehicle only (left flank)
- Control group: test substance (right flank) and vehicle only (left flank)
- Site: posterior right flank
- Concentrations: 25% (w/w)
- Evaluation (hr after challenge): 48 h and 72 h

Additional information on cutaneous exposure
The animals were teated with 0.5 mL sodium lauryl sulfonate (10%, in vaseline) one day prior to epicutaneous induction, in order to induce local skin irritation as the test substance failed to induce skin irritation after cutaneous application in the range finding test.

Challenge controls:

The control group is actually a challenge control.

Positive control substance(s):

yes

Remarks:

mercaptobenzothiazole (June 2002; control: 5 animals, test group: 10 animals; induction: intradermal: 1% (w/w), epicutaneous: 20% (w/w); challenge: 20% (w/w)

Positive control results:

Challenge with 20% mercaptobenzothiazole resulted in skin sensitisation in 10/10 test animals scored with grade 1-3 detected at the 24 and 48 h reading time point. In the control group, no cutaneous reactions were observed.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

induction: 0%; challenge: 25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

induction intradermal: 1%, challenge: 25%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

induction: 0%, challenge: 25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

induction intradermal: 1%, challenge: 25%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

induction intradermal: 1%, challenge: 20%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

weak, mild or moderate sensitisation in 1/10, 4/10 or 5/10 animals, respectively; oedema in 4/10 test animals and crusts in 2/10 animals; dryness of the skin in 5/10 animals

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

induction intradermal: 1%, challenge: 20%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

mild and moderate sensitisation in 4/10 or 6/10 animals, respectively; oedema in 8/10 animals, crusts in 1/10 animals; dryness of the skin in all animals

Remarks on result:

positive indication of skin sensitisation

Results of the range finding test

Intradermal injection of 0.1 - 25% (w/w) test substance induced cutaneous irritation 24 and 48 h after treatment in all animals. Injection of 25% test item solution resulted in necrosis in 1/2 animals detected 48 h after treatment. 6 days after treatment, necrosis was observed in both animals after injection of 5, 10 and 25% test item solution. 6 days after substance injection, crusts were observed in 2/2 animals after injection of 0.1 or 1% test item solution mixed with FCA. Challenge with 40% (w/w) test substance solution induced cutaneous reactions in 1/2 animals (grade 1) 24 h after treatment whereas challenge with 25% test substance solution failed to induce reactions on the skin. In accordance to the criteria for the selection of the concentrations suitable for the main study (please see above under "Details on study design"), concentrations of 1 and 40% (w/w) were chosen for intradermal and epicutaneous induction, respectively, and for challenge 25%.

Results of the main study

Mortality

No mortality or signs of clinical symptoms were recorded until the end of the observation period.

Body weight

Body weight gain was comparable between the test and control group.

Skin reactions

Important local reactions were recorded between Day 6 and Day 16 at the intradermal injection sites of 2 animals of the treated group. After challenge, no cutaneous reactions were observed in control animals. In test animals, a discrete erythema scored with grade 1 was observed on both flanks in 1/20 animals at the 24 hours reading time point. A similar cutaneous reaction was observed in one further animal at the 48 h reading time point on the left flank (vehicle-treated flank). As the cutaneous reactions were determined either on the control and test item treated flank or on the control flank only, the observations were not interpreted as sign of sensitisation. 3/20 test animals revealed dryness of the skin 48 h after patch removal. No other cutaneous reactions were recorded in the test group.

Positive control

Challenge with the positive control substance revealed positive sensitisation reactions in 10/10 test animals at both time points. 48 h after patch removal, weak, mild or moderate reactions were observed on the right flank treated with the positive control substance during induction and challenge in 1/10, 4/10 or 5/10 animals, respectively. In addition, weak or mild sensitisation was observed on the left flank treated with the vehicle during the induction phase in 1/10 and 2/10 animals, respectively. Moreover, oedema and crusts were observed on the right flank treated with the positive control substance during induction and challenge in 4/10 or 2/10 animals, respectively. Dryness of the skin was determined in 5/10 animals. At the later reading time point, mild and moderate skin sensitisation was observed in 4/10 or 6/10 animals, respectively. Weak or mild sensitisation was also observed on the left flank in 2/10 animals each. Moreover, the flank treated wih the positive control during induction and challenge revealed oedema and crusts in 8/10 or 1/10 animals, respectively. Dryness of the skin was observed in 9/10 animals at the second reading time point.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions chosen, the test substance was not a skin sensitizer in guinea pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

 Sensitisation

To evaluate skin sensitising properties of the test substance, data on two skin sensitisation studies according to OECD Guideline 406 are available. The studies have been conducted with the test substance with a content of either 81.8% (solid material) or 22.1% (aqueous solution).

 

In the key study, skin sensitising properties of the test substance (powder with a purity of 81.8%) were evaluated in guinea pigs according to the maximization method of Magnusson and Kligman (OECD 406) in compliance with GLP (M-213501-01-1). Concentrations of 1 and 40% (w/w) were chosen for intradermal and epicutaneous induction, respectively, in regard to the results of a range finding study. The test group comprised 10 male and female Hartley guinea pigs. A control group of 5 male and female animals was included in the study. On Day 1, 3 pairs of intradermal injections were performed in the interscapular region of all animals:

- Freund's complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl (both groups),

- test substance at a concentration of 1% in physiological saline (treated group) or vehicle alone (control group),

- test substance (1%, (w/w)) in a mixture FCA/0.9% NaCl 50/50 (v/v) (treated group) or physiological saline (50%, w/v) in a mixture FCA/0.9% NaCl 50/50 (v/v) (control group).

On Day 7, the skin of animals was treated with 0.5 mL sodium lauryl sulfonate (10%, in vaseline) one day prior to epicutaneous induction, in order to induce local skin irritation. On Day 8, the test substance (treated group) or the vehicle (control group) was applied topically and covered by an occlusive dressing for 48 hours. Challenge with 25% test substance (w/w) occurred on the right flank of all animals on Day 22. The left flank received the vehicle only and served as control. Test substance and vehicle were held in place by an occlusive dressing for 24 hours. After patch removal, no test substance was visible on the skin. No mortality or signs of clinical symptoms were recorded until the end of the observation period and body weight gain was comparable among the groups. No cutaneous reactions were observed in control animals. In test animals, a discrete erythema scored with grade 1 was observed on both flanks in 1/20 animals at the 24 hours reading time point. A similar cutaneous reaction was observed in one further animal at the 48 h reading time point on the left flank. 3/20 test animals revealed dryness of the skin 48 h after patch removal. No other cutaneous reactions were recorded in the test group.

Furthermore, skin sensitisation induced by the test substance was evaluated with a 22.1% aqueous solution following OECD Guideline 406 in compliance with GLP in a supporting study (M-207098-01-1). The test group comprised 10 male and female Dunkin-Hartley guinea pigs. 5 male and female guinea pigs were included in the control group. Concentrations used for induction and challenge were based on a range finding test. On Day 1, 3 pairs of intradermal injections were performed in the interscapular region of all animals:

- Freund's complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl (both groups),

- test substance at a concentration of 25% (corresponding to 5.5% sodium ethyl phosphonate, adjusted for purity) in physiological saline (treated group) or vehicle alone (control group),

- test substance (25%, w/w,corresponding to 5.5% sodium ethyl phosphonate, adjusted for purity) in a mixture FCA/0.9% NaCl 50/50 (v/v) (treated group) or physiological saline (50%, w/v) in a mixture FCA/0.9% NaCl 50/50 (v/v) (control group).

On Day 7, 0.5 mL sodium lauryl sulfonate (10%, in vaseline) were topically applied one day prior to epicutaneous induction, in order to induce local skin irritation as the test substance failed to induce skin irritation after cutaneous application in the range finding test. On Day 8, undiluted test substance (corresponding to 22.1%, treated group) or the vehicle (control group) was applied topically for 48 hours. Challenge with undiluted test substance occurred on Day 22 in control and test animals. Between Day 11 and 22, unexpected mortality occurred in the control (1/10 males) and test group (6/20, 3 animals per sex) accompanied by clinical signs of hypoactivity, piloerection and dyspnea. Similar clinical signs were observed in 2 male test animals (Days 12 or 13 - 21). As these clinical symptoms are known for this species, and mortality occurred in control and test animals late after treatment (Day 11 – 22), the cause of death was most probably not attributed to the treatment. Hence, the mortality was not considered to compromise the validity of the study. After the challenge, no cutaneous reactions were observed in the remaining control animals. In test animals, a discrete erythema (grade 1) was observed in 2/14 animals at the 24 hours reading time point. No other cutaneous reactions were recorded in the test group.

Taken together, the test substance did not induce skin sensitisation in guinea pigs under the experimental conditions chosen.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on the skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.