E17-194T, (P,P-diethyl, monoester with 1,2 propanediol)_

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

Originally, the test was carried out for the purpose of another regulation- registration in the US according to TSCA - US EPA OPPTS 870.240 Acute Eye irritation [EPA712-C-98-195]

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 13, 2017 - January 9, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Originally, the test was carried out for the purpose of another regulation- Registration in the US according to TSCA US EPA OPPTS 870. 2600. [712-C-03-197].

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Principles of method if other than guideline:

none

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:

Reaction mass of 1-hydroxypropan-2-yl diethylphosphinate and 2-hydroxypropyl diethylphosphinate

Cas Number:

2230512-72-6

Molecular formula:

C7H17O3P

IUPAC Name:

Reaction mass of 1-hydroxypropan-2-yl diethylphosphinate and 2-hydroxypropyl diethylphosphinate

Test material form:

liquid

Details on test material:

R&D level

Specific details on test material used for the study:

COA attached

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Animals
Number of Animals: 33
Number of Groups: 9
Number of Animals per Group:
Preliminary Irritation (4 groups): 2 per group
Test (3 groups): 5 per group
Vehicle (Negative) Control: 5
Positive Control: 5

Sex: Female, nulliparous and non-pregnant.
Species/Strain: Mouse, CBAlJ
Age/Body Weight: Preliminary Animals: Young adult (10-11 weeks)
Test and Control Animals: Young adult (11-12 weeks)!
19.7-24.2 grams at experimental start.

Source: Received from Envigo RMS Inc. on November 29, 2017 (preliminary
Irritation Group Animals) and on December 13,2017 (Test and Control Group
Animals).

Housing: The animals were individually housed in plastic solid bottom cages during
the dosing and resting phase of the study. After fmal weighing until sacrifice,
animals were housed in their respective dose groups in plastic cages with bedding.
Bedding in the plastic, solid bottom cages was changed at least once per week.
Enrichment (e.g., nesting material) was placed in each cage. All caging conformed to
the size recommendations in the most recent Guide for the Care and Use of
Laboratory Animals (Natl. Res. Council, 2011).

Animal Room Temperature and Relative Humidity Ranges: 19-23°C and 37-50%,
respectively.
Animal Room Air Changes/Hour: 13. Airflow measurements are evaluated regularly
and the records are kept on file at Product Safety Labs.
Photoperiod: 12-hour light/dark cycle
Acclimation Period: 14 or 21 days
Food: Envigo Teklad Global 16% Protein Rodent Diet@ #2016. The diet was
available ad libitum.
Water: Filtered tap water was supplied ad libitum.
Contaminants: There were no known contaminants reasonably expected to be found
in the food or water at levels which would have interfered with the results of this
study. Analyses of the food and water are conducted regularly and the records are
kept on file at Product Safety Labs

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

The test substance at 25% and 50% w/w mixtures in DMF and the test substance at 100% were selected for test.

No. of animals per dose:

Preliminary Irritation (4 groups): 2 per group
Test (3 groups): 5 per group
Vehicle (Negative) Control: 5
Positive Control: 5

Details on study design:

Preparation of Test Substance
The test substance, as received (neat), was mixed well prior to use. Solubility testing conducted
by PSL indicated that the test substance was soluble in DMF. All preparations were mixed well
prior to dosing.
Preliminary Toxicity Testing
Three test substance concentrations and the vehicle control were used. Test substance
concentrations of 25%, 50% and 100% were tested to determine the highest achievable level that
does not produce overt systemic toxicity or excessive local irritation. Dilutions of the test
substance were prepared as w/w mixtures in DMF. Each group consisted of two mice. The ears
of each mouse were evaluated for erythema and edema pre-dose on Day I and on Days 2, 3, and
6 according to the scoring system described in Table 8.
Twenty-five J.tL of the appropriate concentration of the test substance or the vehicle alone was
applied to the dorsum of both ears of each mouse for three consecutive days. Application was
done using an appropriate size micropipette to accurately deliver 25 ftL. The dose was gently
spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette. No
treatment was made on Days 4 and 5. On Day 6, the sites for each mouse were evaluated for
local reactions (erythema & edema).
Animals were observed daily for signs of toxicity. The Study Director used this data in
conjunction with any pre-existing data to select the three concentrations to be tested. The test
substance at 25% and 50% w/w mixtures in DMF and the test substance at 100% were selected
for test.

Selection of Animals/Dose Levels
Page 9 of44
Study Number 47018
Prior to dosing, the animals were weighed and the ears were checked for any abnormalities or
clinical signs of diseases or injury. Twenty-five healthy naive female mice without pre-existing
ear irritation were selected and distributed (5 mice per group) into the following groups:
Group Purpose Concentration
I Vehicle Control 0%
2 Positive Control Substance 25%HCA
3 Test Substance 25%
4 Test Substance 50%
5 Test Substance 100%
Concentrations were selected based on toxicity, solubility, irritancy, and viscosity.
Sample Preparation
Concentrations of 25%, 50% and 100% were selected for the main test based on results of the
preliminary screening test. Dilutions of the test substance were prepared as w/w mixtures in
DMF. A single concentration of a 25% w/w mixture of HCA in DMF was also prepared. All
dosage preparations were freshly prepared on the day of application.

Test Substance Application
Beginning on Day I, a quantity of 25 ilL of the appropriate test substance concentration, the
positive control substance, or the vehicle alone was applied to the dorsum of both ears of each
mouse once per day for three consecutive days (Days I, 2, and 3) using a micropipette. During
application, the material was gently spread as evenly as possible over the dorsal surface of the ear
using the tip of the pipette.

Dermal Scoring
Prior to each application (Days I, 2, and 3) and on Day 6, the ears were evaluated for erythema
and edema according to the modified Draize scoring system (Draize, Woodard, & Calvary, 1944;
see attached individual dermal irritation score Tables ).

3H-methyl Thymidine Injections
On Day 6 of the study (three days after the final topical application) 250 f1L of sterile phosphate
buffered saline (PBS) containing 20 flCi of 3H-methyl thymidine was injected intravenously via
the tail vein of each mouse.

Lymph Node Assessment
Approximately five hours after the injection, all test and control mice were euthanized via
overdose of inhaled Isoflurane and the draining auricular lymph nodes were excised. The lymph
nodes were evaluated for each individual mouse. A single cell suspension of lymph node cells
(LNC) was prepared in PBS by gently massaging the lymph nodes between the frosted ends of
two microscope slides over a collection vessel. The slides were then rinsed briefly with PBS into
the vessel. The contents of the vessel were transferred to a centrifuge tube and washed with an
excess of PBS and centrifuged for approximately 10 minutes at 1800 rpm, with an RCFl of 489G.
This process was carried out twice. In both cases, the supernatant was decanted and discarded
following each centrifugation. After the second wash, 5 mL of the 5% trichloroacetic acid (TCA)
in distilled water was then added to the sediment and the tube was vortexed briefly. The DNA
was then precipitated in the 5% TCA in distilled water at approximately 4°C overnight
(approximately 18 hours).
Following the overnight precipitation of the DNA, the tubes were centrifuged again for
approximately 10 minutes at 1800 rpm and the supernatant was discarded. The resulting
precipitate was re-suspended using ! mL of the 5% TCA in distilled water and transferred to 10
mL of scintillation fluid. Incorporation of 3H-methyl thymidine was measured by ~-scintillation
counting and expressed as disintegrations per minute, minus background dpm.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was performed. Significance was judged at p < 0.05. The treated groups and
negative vehicle control group were compared using a One-Way Analysis of Variance
(ANOVA), followed by comparison of the treated groups to control by Dunnett's t-test for
multiple comparisons. Where variances are considered significantly different by Bartlett's test,
groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis
of variance followed by Dunn's test) (INS TAT Biostatistics, Graph Pad Software, San Diego,
CA). Outlier analysis was conducted using Grubbs (1969).

Results and discussion

Positive control results:

positive Coutrol - 25% HCA iu DMF: Very slight erythema (score of 1) was
evident at all positive control sites on Days 2 and 3. Well-defined erythema (score of 2) was
evident at all sites on Day 6. Slight edema (score of 1) was present at one site on Day 2, nine
sites on Day 3, and all sites on Day 6. Desquamation was present at all sites on Day 6.

The positive control (HCA) at 25% produced a dernal sensitization response in mice
(SI = 6.02).
see attached table 6- individual DPM values

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

>= 1.08

Test group / Remarks:

25% test substance in DMF

Remarks on result:

other: no indication of skin sensitization

Results 2

Key result

Parameter:

SI

Value:

>= 1.96

Test group / Remarks:

50% test substance in DMF

Remarks on result:

other: no indication of skin sensitization

Results 3

Key result

Parameter:

SI

Value:

>= 2.2

Test group / Remarks:

100% test substance

Remarks on result:

other: no indication of skin sensitization

Any other information on results incl. tables

Clinical Observations

All test, control and preliminary mice were observed for signs of mortality, gross toxicity, and/or

behavioral changes daily (see Tables 2 and 5). Preliminary mice were euthanized via CO2

inhalation and all test and control mice were euthanized via overdose of inhaled Isoflurane

anesthetic on Day 6.

Body Weights

Individual body weights of test and control animals were recorded on Day I (initial) shortly

before test substance application and prior to IV injections on test Day 6.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, the test substance is not considered to be a contact dermal
sensitizer in the LLNA. Proper conduct of the LLNA was conftrmed via a positive response with
25% alpha-Hexylcinnamaldehyde, ~ 95% (HCA), a moderate contact sensitizer.

Executive summary:

A dermal sensitization test was conducted with mice to determine the potential for

E 17-194 T to produce sensitization after repeated topical applications.

Two concentrations (25% and 50%) of the test substance in N,N-dimethylformamide (DMF) and

the neat test substance were topically applied to fifteen healthy test mice (5 mice/group) for three

consecutive days. Three days after the last application, the mice were given an N injection

containing 20 flCi of 3H-methyl thymidine. Approximately five hours later, all animals were

euthanized via an overdose of inhaled Isoflurane and the draining (auricular) lymph nodes were

harvested and prepared for analysis in a scintillation counter. The results are presented in

disintegrations per minute per mouse (dpmlmouse). Each animal's ears were also evaluated for

erythema and edema prior to each application and again on Day 6, prior to the N injection.

A vehicle control group (five animals) and a positive control group (five animals) were

maintained under the same environmental conditions and treated in the same manner as the test

animals. The vehicle control animals were treated with DMF and the positive control group

animals were treated with a 25% w/w mixture of alpha-Hexyicinnamaldehyde (RCA), ::: 95%, in

DMF. In an effort to reduce the total number of animals used, this study was run concurrently

with another study to utilize a common positive control and common vehicle control group.

Based on the results of this study, the test substance is not considered to be a contact dermal

sensitizer in the LLNA. Proper conduct of the LLNA was conftrmed via a positive response with

25% alpha-Hexylcinnamaldehyde, ~ 95% (HCA), a moderate contact sensitizer.