1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

LLNA, Secondary response

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14.07.2014 - 27.10.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

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:

1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

EC Number:

700-453-0

Cas Number:

59802-05-0

Molecular formula:

C26H42O4

IUPAC Name:

1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

HsdWin:NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Nederland, Kreuzelweg 53, 5960 AD Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: 27 - 33 g
- Housing:During the adaptation period up to s·mice were housed together in conventional
Makrolon® type III cages, cages were changed at least twice a week. While during the study period the animals were single-housed in type II cages, cages were changed at least once a week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days
- Indication of any skin lesions: Only healthy animals showing no signs of disease were used in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 oc
- Humidity (%): 40- 70%
- Air changes (per hr): About 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h, with artificial illumination
- IN-LIFE DATES: From: 14.07.2014 To: 14.08.2014

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Based on the results of the dose range-finding studies the following concentrations were
chosen for the main study: induction: 50 %, challenge: 25 %.

No. of animals per dose:

6 animals per group

Details on study design:

PRE-SCREEN TESTS:

In the dose-range-finding study the dorsal part ofboth ears of
the mice were treated on three consecutive days (dl, d2 and d3).
Six animals were placed in each following groups:
Group 1: Vehicle (A/00)
Group 2: 25 % Di-sononyl-terephthalat
Group 3: 50 % Di-isononyl-terephthalat
Group 4: 100 % Di-isononyl-terephthalat
Based on our experiences with this test system and the known properties of the test item the following concentrations were chosen by the sponsor and were used for the dose-range-finding-study: 0% (vehicle control), 25 %, 50% and I 00 %.

- Compound solubility: The test item was formulated immediately before each administration in acetone/olive oil
(4:1) (A/00).
- Irritation: By comparing the specific immunereaction induced by the test item in the
draining lymph nodes (LN cell counts/ LN weights) with the immediate non-specific acute
skin reaction (ear swelling /ear weight) it is possible to discriminate the irritant potential from
the sensitizing potential of the compound tested.
- Systemic toxicity: The body weights of the animals were recorded at the start and the end of the dose-range-finding-study
(day 1 and day 4)
- Ear thickness measurements: Before the first treatment of the ears and before sacrifice the thickness of both auricles of the animals was measured using a spring-loaded micrometer (Oditest, Dyer Company or Fa. Kroeplin). Means, indices and standard deviations
of the ear swelling were calculated by an Excel data sheet.


MAIN STUDY

In the dose-range-finding study the dorsal part of both ears of
the mice were treated on three consecutive days (d1, d2 and d3).
Six animals were placed in each following groups:
Group 1: Vehicle (A/00)
Group 2: 25 % Di-sononyl-terephthalat
Group 3: 50 % Di-isononyl-terephthalat
Group 4: 100 % Di-isononyl-terephthalat
Based on our experiences with this test system and the known properties of the test item the following concentrations were chosen by the sponsor and were used for the dose-range-finding-study: 0% (vehicle control), 25 %, 50% and I 00 %.

A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: three consecutive days ( day 1-3)
- Test groups: 1 test group
Group 7: 50 % Di-isononyl-terephthalat

- Control group: 2 Vehicle groups:
Group 5: Vehicle (A/OO)
Group 6: Vehicle (A/OO)

- Site: epicutaneously onto the pre-shaven left flank


B. CHALLENGE EXPOSURE
- No. of exposures: 3
- Day(s) of challenge: Day 15-17
- Exposure period: This treatment was repeated on three consecutive days
- Test groups: 2
Group 6: 25 % Di-isononyl-terephthalat
Group 7: 25 % Di-isononyl-terephthalat
- Control group: 1
Group 5: Vehicle (A/00)

- Site: dorsal part of both ears
- Evaluation (hr after challenge): 24 hr


ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA/IMDS, Secondary Response
- Criteria used to consider a positive response:
A "positive level", which is 1.4 for cell count indices, has to be exceeded in the dose groups.
Another "positive level'' of ear swelling, which is 2 x 10-2 mm increase, i.e. about 10 % of
the control values, has to be reached in any dose group.
It has to be stated that the "positive levels" mentioned above are exclusively defined for the NMRI outbreed mice used for this study

Positive control substance(s):

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

other: 1-Chloro-2,4-dinitrobenzene (DNCB)

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those
from the control group(s; vehicle) by a one-way analysis of variance (ANOVA) when the
variances are considered homogeneous according to a homogeneity testing like Cochran's
test. Alternatively, if the variances are considered to be heterogenous (p :S 0.05), a
non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance
levels of 5 %. Two sided multiple test procedures were done according to Dunnett or
Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a
probability level of 99 % by Nalimov's method. In addition, for the LLNA/IMDS the smallest
significant differences in the means were calculated by Scheffe's method, which can be used
for both equal and unequal sample sizes.

Results and discussion

Positive control results:

The sensitivity as well as the reliability of the experimental technique is confirmed by
this study.

In vivo (LLNA)

Results

Key result

Parameter:

other: NOEL

Remarks:

The concentration of 50 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization, i.e. induction of specific memory cells.

Value:

>= 50

Remarks on result:

other: These results show that there is no indication for a skin sensitizing effect after administration of 25 % Di-isononyl-terephthalat in this test system as a challenge concentration.

Cellular proliferation data / Observations:

Stimulation indices:
The NMRI mice did not show increases in stimulation indices for cell counts or for weights of
the draining lymph nodes after application ofthe test item
Di-isononyl-terephthalat.
The "positive level", which is 1.4 for the cell count index, was never reached or exceeded in
any dose group.
The "positive level" of ear swelling, which is 2 x 10·2 mm increase, i.e. about 10% of
the control values, has not been reached or exceeded in any dose group.
No substance specific effects were determined for ear weights either.


BODY WEIGHTS
The body weights of the animals were not affected by any treatment.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

These results show that there is no indication for a skin sensitizing effect after administration
of 25 % Di-isononyl-terephthalat in this test system as a challenge concentration.
Therefore, the concentration of 50 % turned out to be the NOEL for the parameters
investigated in this study with respect to skin sensitization, i. e. specific memory induction.