Phosphoric acid, mono- and di-C12-14-alkyl esters

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14th April 2020 - 6th May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The in vitro methods described in OECD 442 C, D & E were found to be unsuitable for use with this substance. This is an UVCB substance, therefore it is not possible to define the molar ratio between substance and peptides for OECD 442 C and therefore OECD 442 C is not applicable in this situation. The substance has a Log P of > 6. For substances with Log P >3.5 a negative result in OECD 442 E cannot be considered in the skin sensitisation assessment. Furthermore, for substances with a Log P between 5 and 7 there are limited validation data available for OECD 442D so a negative result cannot be considered reliable. Evidence from (Q)SARs suggested that the substance is unlikely to possess skin sensitizer properties, however several of the assessed models did not offer convincing statistics and some models failed their applicability domain, therefore no clear conclusion could be formed and the results were not considered sufficient for classification.Since it is not possible to conduct the specific OECD in vitro methods, the assessment of substance sensitisation potential by in vitro means is precluded, an in vivo evaluation was required.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Constituent 2

Reference substance name:

C12-C14 alcohol

IUPAC Name:

C12-C14 alcohol

Constituent 3

Reference substance name:

Mono (C12-C14 alkyl) phosphate ester

IUPAC Name:

Mono (C12-C14 alkyl) phosphate ester

Constituent 4

Reference substance name:

Di (C12-C14 alkyl) diphosphate ester

IUPAC Name:

Di (C12-C14 alkyl) diphosphate ester

Constituent 5

Reference substance name:

Tri (C12-C14 alkyl) triphosphate ester

IUPAC Name:

Tri (C12-C14 alkyl) triphosphate ester

Constituent 6

Reference substance name:

C12-C14 alkyl pyrophosphate esters

IUPAC Name:

C12-C14 alkyl pyrophosphate esters

Constituent 7

Reference substance name:

Ortho-phosphoric acid

IUPAC Name:

Ortho-phosphoric acid

Constituent 8

Reference substance name:

unknown

Molecular formula:

unknown

IUPAC Name:

unknown

Test material form:

solid: bulk

Specific details on test material used for the study:

Batch number: 1023S17201
State: white solid.
Date received: 02 April 2020 and
Storage: at 15-25°C, protected from light.
Purity: 100%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Animal Specifications and Acclimatisation:
Nulliparous, non-pregnant female CBA/CaCrl strain mice were obtained from Charles River (UK) Ltd., Margate. All animals were given a clinical inspection for ill health on arrival and a sample was weighed.
Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. The condition of the animals was assessed daily throughout the acclimatisation period of 8 to 16 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups on the day prior to commencement of treatment.
Animals in the main study were in a body weight range of 19 to 22 g on the day before dosing commenced. Individual body weights were within ±20% of the mean body weight for mice on the study. Based on information from the supplier the mice were approximately 8 to 10 weeks old on Day 1.

Environmental Conditions, Diet and Water:
The animals were kept in the following conditions except for short periods of time where experimental procedures dictated otherwise.

Housing
The animals were housed in groups of up to five during acclimatisation. The preliminary animal was individually housed while the main study animals were housed in pairs from Day-1 in cages that conformed to the ‘Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes' (Home Office, London, 2014).
Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Covance.
No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study.

Water
Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been periodically analysed for specific contaminants.
No contaminants were present in water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Diet
5LF2 EU Rodent Diet 14%, was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.
No contaminants were present in diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Environment
The animal rooms were designed to permit a minimum of 15 air changes per hour. The temperature and humidity ranges were 19 to 25 deg.C and 40 to 70% respectively. Daily recordings of maximum and minimum temperature and humidity were made.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.

Environmental Enrichment
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and nesting materials. The nesting materials were removed from the cages the day before dosing.

Animal Identification and Assignment to Study
A unique number inscribed onto the tail with indelible ink on the day prior to dosing individually identified the mice. A colour-coded card on each cage gave information including study number, animal number and sex.
Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. Overtly healthy animals were arbitrarily allocated to the study groups on the day prior to commencement of treatment.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10, 25 and 50% w/v

No. of animals per dose:

4 in the Main Study;
1 in Preliminary Screening Test.

Details on study design:

Preliminary Screening Test
In the absence of toxicological information regarding irritation and/or systemic toxicity, a preliminary screening test was conducted with one animal.
The mouse was treated by daily application of 25 µL of the substance at the maximum suitable concentration (50% w/v in MEK) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day-1 and prior to termination on Day 6. Both ears were observed for erythema and scored.
Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.

Main Study
Groups of four female mice were assigned to 5 groups (Group 1: vehicle control; Group 2: Substance low concentration (10%w/v); Group 3: Substance intermediate concentration (25% w/v); High concentration (50% w/v);. positive control (25% v/v). Doses were selected from the concentration series 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected on the basis of the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation.

Inlife Procedures
Substance dministration
Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate test or control formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.

Treatment Regimen
The five groups of four female mice were subjected to application of the vehicle control, positive control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3.
On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection. After this treatment, the mice were returned to their cages.
Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exanguination under a deep plane of inhalation anasthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.

Clinical Signs
Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the substance.

Routine Health Checks
All animals were examined at the beginning and end of the working day throughout the acclimatisation and study periods to ensure they were in good health.

Body Weights
Mice were weighed on Day-1 (the day before dosing) and on Day 6 prior to intravenous administration of 3HTdR.

Terminal Procedures
Recovery of Lymph Nodes
Once death had been confirmed each mouse was placed on a bench lined with paper with its ventral aspect uppermost. A mid-line incision from the lower jaw to the sternum was made and the cervical structures were exposed by reflecting the skin. The auricular lymph nodes were located and removed using curved end forceps. Any connective tissue was removed from the capsule of the nodes. The auricular lymph nodes of all mice from each dose group were placed into a petri dish containing 5 mL phosphate buffered saline.

Preparation for Scintillation Count
The lymph nodes collected into each petri dish were cut open and disaggregated by squashing the fragments with a sharp blade. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible.
After 5 minutes the pooled liquor was filtered into a second conical tube by transferring the liquor into a 10 mL syringe and passing it through a stainless steel gauze containing a fabric filter, cut to size (Clarcor UK, Lockertex Filtration Products, Warrington, UK). Any visible sediment remaining prior to filtering was left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes. Following centrifugation, the supernatant was discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes. The supernatant was discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8 deg. C (nominal 4deg. C).
On the following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added.

Scintillation Counting
Once prepared the scintillation vials were placed in the appropriate carrier racks. Two background vials were prepared, one containing ca 10 mL of scintillation fluid and the other containing 1 mL of 5% w/v aqueous trichloroacetic acid and ca 10 mL scintillation fluid. The carrier rack was passed into the scintillation counter. All vials, including the background samples, were submitted for liquid scintillation counting for 10 minutes, using a 3H quench curve.
Incorporation of 3HTdR is measured by ß-scintillation counting as disintegrations per minute (DPM) over a ten-minute period. This value was corrected to account for the background containing 5% w/v aqueous trichloroacetic acid and scintillation fluid.

DATA EVALUATION
The scintillation counter provided data including the DPM value (disintegrations per minute during a ten minute period). The DPM value for each test group was divided by the DPM for the control group to provide the Stimulation Index (SI) value for each test group.

Criteria for Assessment of Test Results
The test result is not valid for those groups producing an SI value of 3.0 or more when the sites of application have shown excessive irritation and for those groups that have shown indications of systemic toxicosis.
The substance is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.
The substance is classified as a non-sensitiser when the maximum value of the SI is less than 3.0. (This result is unchanged by observations of irritation at sites of application of the test formulation).

Positive control substance(s):

other: α-Hexylcinnamaldehyde

Statistics:

N/A

Results and discussion

Positive control results:

Stimulation Index (SI) = 8.41
Disintegrations per minute (DPM) = 14712

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

8.78

Test group / Remarks:

10 %

Remarks on result:

other: Positive

Results 2

Key result

Parameter:

SI

Value:

8.46

Test group / Remarks:

25%

Remarks on result:

other: Positive

Results 3

Key result

Parameter:

SI

Value:

8.9

Test group / Remarks:

50%

Remarks on result:

other: Positive

Cellular proliferation data / Observations:

Preliminary Screening Test
Death or signs of systemic toxicity/excessive irritation were not noted.
Based on this information the dose levels selected for the main test were 10%, 25% and 50% w/v in MEK.
Main Test
Mortality
All animals survived treatment with substance.

Clinical Signs
There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 10, 25 or 50% w/v formulations of the substance.
The vehicle and substance application sites remained free of irritation.
Greasy fur behind the ears was noted on Days 1 to 3 in the Group 2-5 animals and on Days 4 and 5 in the group 5 animals only.

Body Weights
There was no indication of a treatment related effect on body weight.

Stimulation Indices

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls.
The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

Any other information on results incl. tables

Group DPMs and Stimulation Index (SI)

Concentration (% w/v) in MEK	Group Number	Group DPM	Stimulation Index (SI)^a
Vehicle	1	1750	NA
10	2	15358	8.78
25	3	14802	8.46
50	4	15567	8.90
Positive control	5	14712	8.41

a= Stimulation Index of 3.0 or greater indicates a positive result

NA= Not applicable

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The substance is classified as skin sensitizing based on an in vivo skin sensitization study conducted according to OECD TG 429. However, absence of a dose-response and evidence from analogous substances suggests that the result may be a false positive.

Executive summary:

In an in vivo local lymph node assay conducted according to the OECD TG 429 the substance was administered to groups of four female CBA/Ca mice. The treatment concentrations of the substance were chosen from a pre-screen test which showed sensitizing potential at 50 % w/v of the substance. The substance was applied onto the ear at concentrations of 0, 10, 25 or 50% w/v on three consecutive days. Three days after the last exposure all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal and scintillation counting (incorporated radioactivity) to determine cellular proliferation was performed.

The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. Mean DPM/animal values for the experimental groups treated with substance concentrations 10, 25 and 50% were 15358, 14802 and 15567 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1750 DPM. The SI values calculated for the substance concentrations of 10, 25 and 50% were 8.78, 8.46 and 8.90, respectively. Results showed that there is a potential of skin sensitisation in all treatment groups. No mortality occurred and no clinical signs related to systemic toxicity were observed in any of the animals. Body weights and body weight gain of animals remained in the same range as controls over the study period. Therefore, the substance is considered to be category 1 skin sensitizing based on GHS criteria. However, absence of a dose-response and evidence from analogous substances suggests that the result may be a false positive.