Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-[2-[bis(phosphonomethyl)amino]methylethyl]-.omega.-[2-[bis(phosphonomethyl)amino]methylethoxy]-, sodium salt (1:?)

Endpoint summary

Description of key information

No in vitro or in chemico data were submitted for this substance because adequate data from an in vivo skin sensitisation study are available (carried out before 11 October 2016).

Skin Sensitisation: Non-sensitiser; OECD 406; Arcelin (2012)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 07-Jul-2011, Experimental Completion Date: 12-Aug-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

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)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Existing data using the GPMT method were available prior to the LLNA becoming the preferred method for skin sensitisation testing.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF (Breeder: Harlan Laboratories B.V., The Netherlands).
- Age at study initiation: 4 weeks
- Weight at study initiation:
Bodweight at pretest start: Pretest Groups 325.9 - 351.7 g
Body weight at beginning of acclimatisation period: Test and control groups: 321.3 - 361.5 g
- Housing: In groups of up to 10 animals in stainless steel cages with standard softwood bedding
- Diet (e.g. ad libitum): Teklad Global Guinea pig diet 2040C, ad libitum
- Water (e.g. ad libitum): Community tap water in bottles, ad libitum.
- Acclimation period: Twelve days for the test and control groups of the main test under standard laboratory conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): continuously monitored environment with a room temperature of 22 ± 3 °C
- Humidity (%): relative humidity between 30-70%,
- Air changes (per hr): Air-conditioned with 10-15 air changes per hour.
- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 hours light and 12 hours dark

Route:

intradermal and epicutaneous

Vehicle:

water

Remarks:

(see details on study design for justification).

Concentration / amount:

Main Study:
Intradermal induction: 10% dilution of the test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline.
Epidermal induction: 75% in purified water
Challenge: 75% in purified water and purified water alone

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

(see details on study design for justification).

Concentration / amount:

Main Study:
Intradermal induction: 10% dilution of the test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline.
Epidermal induction: 75% in purified water
Challenge: 75% in purified water and purified water alone

No. of animals per dose:

Main study:
Test group: 10 males
Control group: 5 males

Details on study design:

VEHICLE:
The vehicle was selected based on preliminary solubility testing, which was performed before the study initiation date. The test item was prepared at a concentration of 75% (w/w) in purified water, which was well soluble and resulted in a viscous yellow liquid. Therefore, this concentration was used as highest concentration in the epidermal pretest.

For the intradermal pretest, the test item was prepared at a concentration of 25% in FCA/physiological saline. This preparation went through a needle of 0.5 x 16 mm. Therefore, this test item concentration in purified water was used as the highest concentration in the intradermal pretest.

AUXILIARY COMPOUNDS:
FCA - Freund's Adjuvant - complete
Physiological Saline - Natrium chloratum 0.9%

PREPARATION OF DOSE FORMULATIONS:
The test item and vehicle or auxiliary compound were placed into a glass beaker on a tared Mettler balance and weight/weight dilutions were prepared. A magnetic stirrer and an ultraturrax was used to ensure homogeneous distribution of the test item preparation. The preparations were made immediately prior to each dosing.
Homogeneity of the test item formulation was maintained during administration using a magnetic stirrer.

SELECTION OF TEST ITEM CONCENTRATION FOR MAIN STUDY:
Intradermal Induction:
The test item prepared at 10% (used for the intradermal induction) was the highest tested concentration causing mild skin irritation while the test item concentrations of 25% in purified water did cause a moderate irritation (with blanching) during the pretest. Therefore, the test item concentration of 10% in purified water was considered to be the suitable concentration selected for the main intradermal induction.

Epidermal Induction:
The test item prepared at 75% (used for the epidermal induction) was the highest technically applicable concentration and did not cause any skin irritation during the pretest. This concentration was considered to be appropriate for the main epidermal induction.

Epidermal Challenge
The test item prepared at 75% (used for the epidermal induction) was the highest technically applicable concentration and did not cause any skin irritation during the pretest. This concentration was considered to be appropriate for the challenge in the main test.

To determine the different concentrations, intradermal and epidermal pretests were performed as described below.

RATIONALE:
The application form and dose was used to detect a possible allergenic potential of the test item applied.

OBSERVATIONS:
Viability / Mortality: Daily from delivery of the animals to the termination of the test.
Clinical Signs: Daily from delivery of the animals to the termination of test.
Local signs: Skin responses were graded during the pretest, induction and challenge period
Body Weights: At delivery/acclimatization start, at test item treatment day in the pretest, at the end of the pretest, at test day 1 (day of treatment) and at the termination of the study.

PATHOLOGY:
Necropsy:
No necropsy was performed on all surviving animals. The control and test animals were euthanized at the end of the test period by intraperitoneal injection of pentobarbitone at a dose of 2.0 mL/kg of 162 mg/mL sodium pentobarbitone and discarded. The pretest animals were euthanized as described at the treatment start of the main study.

TREATMENT METHOD:
The animal's fur was shaved with a fine clipper blade just prior to exposure. Intradermal injections or closed patches were applied to the animals as follows:

0.1 mL/site for the intradermal administrations or 0.2 to 0.3 mL or 0.2 to 0.3 g on a patch of filter paper for the epidermal administrations.

The patch was covered by a strip of aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for 24 (epidermal pretest and epidermal challenge) or 48 hours (epidermal induction).

Identical patching method was used for the epidermal pretest, epidermal induction and epidermal challenge.

PRETEST:
The pretest was performed during the acclimatization period of the animals for the main test.
The test item concentrations described below were selected during a preliminary solubility testing which was performed before the study initiation date.

Intradermal injections:
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig (no. 55). Five days later, three intradermal injections (0.1 mL/site) were made into the clipped flank of the same guinea pig at concentrations of A = 25%, B = 10% and C = 5% of the test item in purified water.

Dermal reactions were assessed 24 hours later.

Based on the results, a test item concentration of 10% was selected for intradermal induction in the main test.

Epidermal Applications:
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs (nos. 56 - 57). Five days later, four patches of filter paper (3 x 3 cm) were saturated with the test item at D = 75% (technically the highest possible concentration to be applied sufficiently), E = 50%, F = 25% and G = 10% in purified water and applied to the shaved flanks of the same guinea pigs. The volume of test item preparation applied was approximately 0.2 mL for the test item concentrations of 50%, 25% and 10% and an amount of approximately 0.2 g was applied for the test item concentration of 75%. The occlusive dressings were left in place for 24 hours.

The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

The allocation of the different test item dilutions to the sites (D, E, F, G) on the two animals was alternated in order to minimize site-to-site variation in responsiveness.

Based on the results the test item concentration selected for epidermal induction and challenge in the main test was 75% in purified water.

MAIN STUDY:
INDUCTION:
Intradermal injection performed on Test Day 1:

An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made just within the boundaries of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test item at 10% in purified water.
3) The test item at 10% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epidermal induction performed on Test Day 8:
On test day 7, approximately 24 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) of the animals of the test and control groups was clipped, shaved free of hair and the test area was pretreated with 0.5 mL of 10% Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10% concentration of SLS enhances sensitisation by provoking a mild inflammatory reaction.

On test day 8 the scapular area (approximately 6 x 8 cm) was again shaved prior to epidermal induction. A 2 x 4 cm patch of filter paper was saturated with the test item at 75% in purified water and placed over the injection sites of the test animals. The amount of test item preparation applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.

The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

CHALLENGE
Performed on Test Day 22

The test and control guinea pigs were challenged two weeks after epidermal induction and treated in the same way.

Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested nonirritating concentration of 75% in purified water (applied to the left flank) and the vehicle only (purified water applied to the right flank) using the same method as for the epidermal application.
The amount of test item preparation applied was 0.2 g and a volume of approximately 0.2 mL was used for the vehicle. The dressings were left in place for 24 hours.

The scoring method used for the pretest, induction and epidermal challenge was identical. It was performed 24 ± 2 hours after removal of the dressings for the intradermal and epidermal pretest, induction and challenge and repeated 24 ± 2 hours later (48-hour grades) for the epidermal pretest, epidermal induction and challenge.

Challenge controls:

Induction:

Intradermal:
Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Purified water.
3) 1:1 (w/w) mixture of purified water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epidermal:
The guinea pigs were treated as for the test group with purified water only, applied at a volume of approximately 0.3 mL.

Challenge:
The test and control guinea pigs were challenged two weeks after epidermal induction and treated in the same way.

Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested nonirritating concentration of 75% in purified water (applied to the left flank) and the vehicle only (purified water applied to the right flank) using the same method as for the epidermal application.
The amount of test item preparation applied was 0.2 g and a volume of approximately 0.2 mL was used for the vehicle. The dressings were left in place for 24 hours.

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamaldehyde

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of alpha-hexylcinnamaldehyde which is recommended by the OECD 406 Guidelines and is known to have moderate skin sensitisation properties in the guinea pig strain. The results from the positive control test confirm alpha-hexylcinnamaldehyde as skin sensitizer, as it produced allergic contact dermatitis in >30% of the test animals.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75% (test item in purified water)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75% (test item in purified water). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75% (test item in purified water)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75% (test item in purified water). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Purified water only

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Purified water only. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Purified water only

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Purified water only. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

other: Control group

Dose level:

75% (test item in purified water)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group. Dose level: 75% (test item in purified water). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

other: Control group

Dose level:

75% (test item in purified water)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group. Dose level: 75% (test item in purified water). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Purified water only

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group. Dose level: Purified water only. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Purified water only

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group. Dose level: Purified water only. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Observations:

Viability / Mortality / Macroscopic Findings

No intercurrent deaths occurred during the course of the study, hence no necropsies were performed.

Clinical Signs:

No clinical signs were recorded throughout the entire observation period.

Body Weights:

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Skin Reactions in the Intradermal Induction:

Expected common findings were observed in the test and control groups after the different injections using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

No detailed description of the skin reactions is given in the report as these FCA effects are well known.

Skin Reactions in the Epidermal Induction:

See Tables 1 to 2 (attached background material).

Control Group

All 5 control animals were observed with discrete/patchy erythema at the 24- and 48-hour reading. Additionally, several animals (4 at the 24 hours and 2 at the 48 hours) were also observed with skin desquamation.

Test Group

All 10 test animals were observed with discrete/patchy erythema at the 24- and 48-hour reading after treatment with the test item prepared at 75% in purified water. Additionally, 2 test animals at the 24 hours and 4 test animals at the 48 hours were observed with skin desquamation.

The reactions observed in both groups occurred following pretreatment with 10% SLS in paraffinum perliquidum.

Skin Reactions in the Challenge:

See Tables 3 to 6 (attached background material)

Control Group

No local skin reactions were observed in the control animals when treated with purified water alone or when treated with the test item prepared at 75% in purified water.

Test Group

No local skin reactions were observed in the test animals when treated with purified water alone or when treated with the test item prepared at 75% in purified water.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the conducted sensitisation test (M&K-test) in guinea pigs the test item does not have to be classified and labeled as a skin sensitizer.

Executive summary:

In order to assess the cutaneous allergenic potential of the test item, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6.

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 10% dilution of the test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 75% in purified water one week after the intradermal induction and following pretreatment of the test areas with 10% Sodium-Lauryl-Sulfate (SLS) approximately 24 hours prior to application of the test item. The animals of the control group were intradermally induced with purified water and FCA/physiological saline and epidermally induced with purified water under occlusion following pretreatment with 10% SLS.

Two weeks after epidermal induction the test and control animals were challenged by epidermal application of the test item at 75% in purified water and purified water alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

No intercurrent deaths occurred during the course of the study.  No toxic signs were evident in the guinea pigs of the control or test group. No local skin effects were observed in the guinea pigs of the control or test group.

Based on the above mentioned findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, the test item is not considered a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) 1907/2006 as amended: Annex VII, section 8.3: the in chemico or in vitro / ex vivo study does not need to be conducted based on existing data sufficient for classification and labelling being already available. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.3, October 2015) the study does not need to be conducted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In accordance with REACH Regulation (EC) 1907/2006 as amended: Annex VII, section 8.3: the in chemico or in vitro / ex vivo study does not need to be conducted based on existing data sufficient for classification and labelling being already available. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.3, October 2015) the study does not need to be conducted.

Skin Sensitisation - in vivo

In order to assess the cutaneous allergenic potential of the test item, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6.

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 10% dilution of the test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 75% in purified water one week after the intradermal induction and following pretreatment of the test areas with 10% Sodium-Lauryl-Sulfate (SLS) approximately 24 hours prior to application of the test item. The animals of the control group were intradermally induced with purified water and FCA/physiological saline and epidermally induced with purified water under occlusion following pretreatment with 10% SLS.

Two weeks after epidermal induction the test and control animals were challenged by epidermal application of the test item at 75% in purified water and purified water alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

No intercurrent deaths occurred during the course of the study.  No toxic signs were evident in the guinea pigs of the control or test group. No local skin effects were observed in the guinea pigs of the control or test group.

Based on the above mentioned findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, the test item is not considered a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

There is no evidence available to suggest that the substance will be a respiratory sensitiser.

Justification for classification or non-classification

The substance does not meet the criteria for classification as a sensitiser in accordance with Regulation (EC) 1272/2008 (CLP).