Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

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)

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to conditions described in Column 2 of Section 8.5.3 of Annex VIII testing by the dermal route does not need to be conducted if: (1) the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and (2) no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation). Since the substance does not meet the criteria for classification as acute toxicity by the oral rout (LD50 > 2000 mg/kg bw). No signs of systemic toxicity were observed in an in vivo study with dermal exposure (guinea pig maximisation test), it is therefore unlikely that the acute dermal toxicity will exceed the oral toxicity.
Since this substance does not meet the criteria for classification as acute toxicity by the oral route and no systemic effects have been observed in an in vivo study with dermal exposure, testing by the dermal route for the substance is considered scientifically not justified.

See Cross references for relevant supporting toxicological data.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 2019 - 03 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines (2000), including the most recent revisions

Version / remarks:

2000

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: Crl: WI(Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 10-11 weeks old)
- Weight at study initiation: 182 to 207 g
- Fasting period before study: Animals were deprived of food overnight (for a maximum of 20 hours) prior to dosing and until 3-4 hours after administration of the test item. Water was available.
- Housing: Animals were group housed (up to 3 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to municipal tap-water
- Acclimation period: At least 5 days
- Method of randomisation in assigning animals to test and control groups: Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 50 to 53
- Air changes (per hr): ten or greater
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 November 2019 To: 03 December 2019

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: The dose volume for each animal was based on the body weight measurement prior to dosing. Dose volume (mL/kg body weight) was calculated as follows: Dose level (g/kg) / specific gravity or density (g/mL) * purity correction factor.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: The toxicity of the test item was assessed by stepwise treatment of groups of 3 females. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines. The onset, duration and severity of the signs of toxicity were taken into account for determination of the time interval between the dose groups. The first group was treated at a dose level of 2000 mg/kg. Based on the results, one additional group was dosed at 2000 mg/kg.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality/Moribundity Checks:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Clinical Observations:
Post-dose observations were performed at periodic intervals on the day of dosing (at least three times) and once daily thereafter. All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate).
Body Weights:
Animals were weighed individually on Day 1 (pre-dose), 8 and 15. A fasted weight was recorded on the day of dosing. Terminal body weights were collected from animals found dead or euthanized moribund after Day 1.
- Necropsy of survivors performed: All animals surviving to the end of the observation period were sacrificed by oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded.

Statistics:

Not performed.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

One female was found dead on Day 9. No further mortality occurred.

Clinical signs:

other: Hunched posture was noted for all animals between Days 1 and 11 and piloerection was noted for all animals between Days 1 and 10. Salivation was noted for three out of six animals on Day 1.

Gross pathology:

Abnormalities of the thymus (reduced in size) and the forestomach (irregular surface) were found in the animal that died during the study, at macroscopic post mortem examination. Macroscopic examination of the surviving animals at termination did not reveal any abnormalities.

Interpretation of results:

other: Not classified.

Remarks:

According to Regulation (EC) No. 1272/2008.

Conclusions:

In an acute oral toxicity study with female rats, performed according to OECD 423 test guideline and GLP principles, a LD50 >2000 mg/kg bw was determined.

Executive summary:

The substance was administered by oral gavage to two consecutive groups of three female Wistar Han rats at 2000 mg/kg body weight, performed according to OECD 423 test guideline and GLP principles.

One female was found dead on Day 9. No further mortality occurred. Hunched posture was noted for all animals between Days 1 and 11 and piloerection was noted for all animals between Days 1 and 10. Salivation was noted for three out of six animals on Day 1. In the animal that died during the study, body weight loss was seen. Body weight loss was noted in one surviving animal and reduced body weight gain was noted for the majority of surviving animals. Abnormalities of the thymus (reduced in size) and the forestomach (irregular surface) were found in the animal that died during the study, at macroscopic post mortem examination. Macroscopic examination of the surviving animals at termination did not reveal any abnormalities.

Based on the results, a LD50 >2000 mg/kg bw was determined and the substance does not have to be classified for acute oral toxicity according to Regulation (EC) No. 1272/2008.

Cross-reference 2

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:

01 October 2019 - 29 November 2019

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:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines (2000), including the most recent revisions.

Version / remarks:

2000

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Dunkin Hartley guinea pig was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals are based on the test guidelines.
The guinea pig Maximization test was selected since the test item 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
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 5 weeks old)
- Weight at study initiation: 276 to 334 g
- Housing: Animals were group housed (up to 5 animals of the same sex and same dosing group together) in labelled Noryl cages containing sterilized sawdust as bedding material.
- Diet: Free access to complete maintenance diet for guinea pigs (MS-H, SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum, except during designated procedures. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): Ten or greater
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 08 October 2019 To: 29 November 2019

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

1% for the intradermal induction and 5% for the epidermal induction.

Day(s)/duration:

Intradermal induction: 7 days. Epidermal induction: 48 hours.

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

5% for the challenge phase.

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test animals: 10
Control animals: 5

Details on study design:

RANGE FINDING TESTS:
Series of test item 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% (undiluted), 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 eight animals selected were 4 weeks old. No body weights were determined.
Intradermal injections:
Initially, a series of four test item 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 hours 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 item concentrations was tested; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) 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 hours, the dressing was removed and the skin cleaned of residual test item using water.
The resulting dermal reactions were assessed for irritation 24 and 48 hours after removal of the dressings.
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.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
1) Intradermal injections on day 1:
- Site: scapular region. One of each pair was on each side of the midline and from cranial to caudal:
Three pairs of intradermal injections:
1) 0.1 mL: FCA (50% in water for injection)
2) 0.1 mL: test substance at a 1% concentration (control animals: 0.1 mL corn oil)
3) 0.1 mL: 1:1 mixture of the test substance at a 2% concentration + FCA (undiluted)
- Readings: on day 3 (48 hrs after the injections)

2) Topical application on day 8:
- Amount: 0.5 mL (control animals: 0.5 mL corn oil) 5% test substance
- Area: approximately 6 cm^2
- Exposure period: 48 hours (occlusive)
- Readings: scores were rated directly after patch removal

B. CHALLENGE EXPOSURE (all animals, with the 5% test substance and the vehicle)
- Day of challenge: day 21
- Exposure period: 24 hours (occlusive)
- Site: flank
- Amount: 0.1 mL
- Readings: scores were rated 24 and 48 hours after patch removal

OBSERVATIONS
Mortality/Moribundity Checks: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
Toxicity: At least once daily.
Body weights: Animals were weighed individually on Day 1 (pre-dose) and after each challenge on Day 24.
Necropsy: No necropsy was performed.
Irritation: Skin reactions were graded according to OECD 406. The intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis. A description of all other local effects was recorded.

Challenge controls:

Not applicable.

Positive control substance(s):

yes

Remarks:

the results of the latest reliability check, performed in June 2019 with Alpha-Hexylcinnamaldehyde, are reported.

Positive control results:

The latest reliability check shows a sensitisation rate of 100%.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

vehicle

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:

test chemical

Dose level:

5%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

vehicle

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

Dose level:

50% Alpha- Hexylcinnamaldehyde, technical grade

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50% Alpha- Hexylcinnamaldehyde, technical grade

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

other: Not classified.

Remarks:

According to Regulation (EC) 1272/2008.

Conclusions:

In a guinea pig maximisation test method the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and GLP principles, showing a sensitization rate of 0 per cent.

Executive summary:

In a guinea pig maximisation test method the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and GLP principles.

Slight to moderate erythema was observed during the intradermal induction (conc. 1%) at the injection sites in control and test animals. Slight erythema was observed following the epidermal induction (conc. 5%) in 8 test animals. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

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

There was no evidence that the substance had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 5% test item concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

Based on these results the substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).