Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters

Molecular formula:

unspecified

IUPAC Name:

Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters

Test material form:

liquid: viscous

Details on test material:

- Name (as cited in the report): SAT 150011
- Chemical name: Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters
- Appearance: clear, light amber liquid
- Purity: 70.2% mono and diesters
- Batch No.: RP GW 915 MU/11.06.2015
- Expiry date: 01/06/2018
- Date of receipt : 07/07/2016
- Storage conditions : ambient (15-25 °C)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Species/strain: healthy CBA/CaOlaHsD mice
- Source: Envigo, 5800 AN Venray, The Netherlands
- Sex: female (nulliparous and non-pregnant)
- Age at the beginning of the study: 9-10 weeks
- Number of animals: 5 mice / group, 15 mice / prescreen test
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals are bred for experimental purposes.
The animals were randomly selected. Identification was ensured by cage number and individual marking (tail).

HOUSING AND FEEDING CONDITIONS
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

6.25, 12.5 and 25%

No. of animals per dose:

5

Details on study design:

The LLNA has been developed as an alternative method for the identification of skin sensitising test items and measures the proliferation of lymphocytes isolated from lymph nodes (auricular lymph nodes) draining the site of exposure (dorsal aspect of the ears) in mice. Lymphocyte proliferation is measured by determining the incorporation of 3H-methyl thymidine.

Positive control substance(s):

other: Periodical reliability check with p-phenylenediamine (CAS 106-50-3)

Results and discussion

Positive control results:

RELIABILITY CHECK (mean values):

Negative Control (vehicle):
- CPM 1047.0
- DPM 2360.2
- SI 1.0

Positive Control (p-phenylenediamine):
- CPM 5945.6
- DPM 13171.2
- SI 5.6

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

9.9

Results 2

Parameter:

SI

Value:

1.6

Test group / Remarks:

6.25%

Results 3

Parameter:

SI

Value:

4

Test group / Remarks:

12.5%

Results 4

Parameter:

SI

Value:

5.3

Test group / Remarks:

25%

Any other information on results incl. tables

PRESCREEN TEST

Both animals treated with the undiluted test item as well as 1/4 animals (animal no. 6) treated with the test item at a concentration of 50% in AOO showed signs of excessive irritation (increased ear thickness >= 25%) at both application sites (mean) at least on day 6. In addition 1/4 animals (animal no. 8) treated with the test ltem at a concentration of 25% in AOO showed signs of excessive irritation on the right ear and only on day 3. The remaining animals treated with the test item at a concentration of 50% or 25% in AOO showed at most slight signs of irritation, i.e, increased ear thickness, at both application sites on day 6. In addition animals treated with 100% and 50% of test item showed slight erythema, i.e. erythema grade 1.

On day 6, no signs of irritation were detected in any of the animals treated with the test item at a concentration of 12.5% as well as with the negative control.

All animals, but animal 4 and 7 (both from 50% prescreen test group), showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test. The stronger body weight decrease in animals 4 and 7 is considered to be a moderate to severe sign of systemic toxicity in this study design. Despite of that no signs of systemic toxicity were recorded.

The concentrations chosen for the main study were based on systemic toxicity and excessive local skin irritation data from the prescreen, as shown above. Both effects should be excluded in the main test. After dosing of 25% test item in AOO only 1/4 animals showed severe irritation assessed by ear thickness increase on one single ear and only on day 3 and not on day 6. Therefore it was concluded that those findings represent the highest applicable concentration for the main test which is at the boarder of severe irritation. All other groups of the main test were administered with test item diluted by factor of 2, i.e. 12.5% and 6.25% in AOO.

MAIN STUDY

Two of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration of 6.25% was 1.6

The stimulation index at a concentration of 12.5% was 4.0

The stimulation index at a concentration of 25.0% was 5.3

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

All animals survived throughout the test period showing clinical signs.

Group	Animal No.	Weight change [g]	Systemic Effects	Local Effects
6.25%	1	-1	no specific findings	no specific findings
	2	1
	3	0
	4	1
	5	1
12.5%	6	1	day 1, 2, 4, 5, 6: no specific findings day 3: Erythema grade 1, wet fur	day 1, 2, 4, 5, 6: no specific findings day 3: Erythema grade 1, wet fur
	7	-1
	8	1
	9	1
	10	0
25%	11	1	day 1: no specific findings day 2 -5: wet fur day 3: Erythema grade 1 day 6: Eschar (2 animals)	day 1: no specific findings day 2 -5: wet fur day 3: Erythema grade 1 day 6: Eschar (2 animals)
	12	1
	13	3
	14	2
	15	1
Control	101	0	no specific findings	no specific findings
	102	0
	103	0
	104	2
	105	2

The means of the ear thickness per group showed relevant difference compared to the negative control due to excessive irritation at both appflcaflon sites in one animal treated with the highest dose on day 6 (ear thickness increase > 25%). Animal no. 13 and 15 (both from 25% group) showed an increase of ear thickness on day 6 of more than 25% which is considered to be excessive. Those animals were excluded from calculations of the stimulation index.

Mean ear thickness	day 1	day 3	day 6
6.25%	0.16 mm	0.16 mm	0.16 mm
12.5%	0.16 mm	0.18 mm	0.17 mm
25%	0.16 mm	0.18 mm	0.20 mm
negative control	0.17 mm	0.17 mm	0.17 mm

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 9.9%.
The mean values of the ear thickness per group showed no relevant difference compared to day 1. However, animals no 13 and 15 (25%) showed a relevant difference in ear thickness (increase > 25% on day 6) which is considered to be a sign of excessive irritation. Therefore, results from these animals were excluded from calculation of the stimulation index.
Consequently, according to OECD 429, solutions or preparations containing more than 9.9% Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters are expected to have a stimulation index of >3, and are therefore considered to be dermal sensitisers.
According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item Phosphoric acid, mono C16-20 (branched, even numbered) alkyl esters has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Executive summary:

SUMMARY

On the basis of the test results given below and in conformity with the criteria given in Commission Regulation (EU) No 286/2011 the substance should be:

- classified into sub-category 1B

On the basis of the test results given below and in conformity with the criteria given in GHS (Globally Harmonized Classification System) the substance should be:

- classified into sub-category 1B

Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 6.25%, 12.5% and 25% (v/v), each diluted with AOO 4:1 (v/v).

At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed.

Species/strain: Mice, CBA/CaOlaHsd

Number of animals: 20/main test

Vehicle: AOO (4:1 (v/v) acetone/olive oil)

Two of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration of 6.25% was 1.6

The stimulation index at a concentration of 12.5% was 4.0

The stimulation index at a concentration of 25.0% was 5.3

Due to excessive irritations, with an ear thickness increase of more than 25% on day 6, values from animals no 13 and 15 were excluded from calculation of the stimulation index.

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 9.9%.