Phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2'-iminobis[ethanol]

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation)

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 11, 2017 to Apr. 03, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, part B. “Skin Sensitization: Guinea-Pig Maximization Test (GPMT)”

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Guinea Pig Maximization Test was selected since the test substance is a surfactant and the Local Lymph Node Assay as preferred alternative has shown to provide false positive results for surfactants

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France; Charles River Deutschland, Sulzfeld, Germany
- Age at the Initiation of Dosing: Young adult animals (approximately 4 weeks old).
- Weight at the Initiation of Dosing: 258 to 289 g.
- Acclimation: 5 d before the commencement of dosing.
- Housing: Up to 5 animals of the same sex and same dosing group together in labeled Noryl cages containing sterilized sawdust as bedding material
- Diet: Complete maintenance diet for guinea pigs (MS-H, SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
- Water: Municipal tap-water was freely available to each animal via water bottles.
- Other: Nulliparous and non-pregnant animals were included in the study

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24°C
- Humidity: 40 to 70%
- Air changes: Ten or greater air changes per hour with 100% fresh air
- Photoperiod: 12 hour light/12 hour dark cycle

ANIMAL ENRICHMENT: For psychological/environmental enrichment, animals were provided with shelters, Play tunnels, Datesand, except when interrupted by study procedures/activities.

No. of animals per dose:

Experimental group: 10 females
Control group: 5 females

Details on study design:

VEHICLE: Water (Elix, Millipore S.A.S., Molsheim, France).

PREPARATION OF TEST SUBSTANCE
Test substance dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
Any residual volumes were discarded.
Analysis of test substance in vehicle for concentration, stability, homogeneity was not performed.

ANIMAL SELECTION, IDENTIFICATION AND ASSIGNMENT TO EXPERIMENTAL GROUPS
- Animal Identification: At study assignment, each animal was identified using an ear tattoo.
- Assignment to experimental groups: Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
- Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. The selection of concentrations was based on the following criteria:
- The concentrations are well-tolerated systemically by the animals.
- For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
- For challenge exposure: the maximum non-irritant concentration.
Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100%, 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The six animals selected were between 4 and 9 weeks old. No body weights were determined.

- Intradermal injections:
Initially, a series of four test substance concentrations was tested; the highest concentration was the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 h after treatment.
Based on the results in the initially treated animals, two additional animals were treated in a similar manner with four lower concentrations at a later stage.

- Epidermal application:
A series of four test substance concentrations was tested; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each or an equivalent amount when dosed with a spatula) per animal to the clipped flank, using Metalline patches# (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape and subsequently Coban elastic bandage. The initially used animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations. After 24 h, the dressing was removed and the skin cleaned of residual test substance using water.
The resulting dermal reactions were assessed for irritation 24 and 48 h after removal of the dressings.

MAIN STUDY
The concentrations and induction method were selected based on the results of the preliminary irritation study.

A. INDUCTION EXPOSURE (Experimental animals)
- Day 1: The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
a) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Sigma-Aldrich, Steinheim, Germany) with water for injection (Fresenius AG, Bad Homburg, Germany).
b) The test substance at a 2% concentration.
c) A 1:1 w/w mixture of the test substance, at twice the concentration used in (b) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial a) to caudal c).
- Day 3: The dermal reactions caused by the intradermal injections were assessed for irritation.
- Day 7: The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.
- Day 8: The 10% SDS treated area between the injection sites was treated with 0.5 mL or an equivalent amount when dosed with a spatula of a 100% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
- CONTROL ANIMALS: The control animals were treated as described for the experimental animals except that, instead of the test substance, the vehicle was administered.

B. CHALLENGE EXPOSURE (Experimental and Control animals)
- Day 21: One flank of all animals was clipped and treated by epidermal application of a 100% test substance concentration and the vehicle (0.1 mL each, using Patch Test Plasters (Curatest F®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
- The dressing was removed after 24 h exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 h after removal of the dressing.

TERMINATION
- After termination, animals were sacrificed using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and an intra-peritoneal injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Challenge controls:

Control animals were treated similar to test animals in 'CHALLENGE EXPOSURE'. For details refer to the section 'Details on study design'

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% test substance concentration

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were noted for any animal 24 h after exposure

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% test substance concentration

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were noted for any animal 48 h after exposure

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% test substance concentration

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% test substance

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

not measured/tested

PRELIMINARY IRRITATION STUDY

Based on the results, the test substance concentrations selected for the main study were a 2% concentration for the intradermal induction and a 100% concentration for the epidermal induction exposure. Only very slight erythema was observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals of the main study were treated with 10% SDS approximately 24 h before the epidermal induction, to provoke a mild inflammatory reaction.

A 100% test substance concentration was selected for the challenge phase.

MAIN STUDY

INDUCTION PHASE: The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

CHALLENGE PHASE: No skin reactions were evident after the challenge exposure in the experimental and control animals.

TOXICITY / MORTALITY: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

There was no evidence that test substance caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 100% test substance concentration in the challenge phase.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, test substance was determined to be non-sensitising.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the Guinea-Pig Maximisation Test method (GPMT) according to OECD Guideline 406, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, 10 experimental animals were intradermally injected with a 2% concentration and epidermally exposed to a 100% concentration (undiluted). Five control animals were similarly treated, but with vehicle alone. Approximately 24 h before the epidermal induction exposure, all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were epidermally challenged with undiluted test substance (100% concentration) and the vehicle. There was no evidence that the test substance caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental or control animals in response to a 100% test substance concentration in the challenge phase. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. Under the study conditions, the test substance was determined to be non-sensitising (Van Sas, 2018).