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

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08.11.2010 to 22.12.2010

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study design is not appropriate for the test item.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation:eight to twelve weeks of age
- Weight at study initiation: 17.5 to 20.1 g
- Housing: The animals were allocated without conscious bias to cages within the treatment groups. They were housed two animals, in solid bottomed polycarbonate cages with a stainless steel mesh lid.
- Diet :ad libitum
- Water: ad libitum
- Acclimation period:at least 5 days prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes: The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours continuous dark per 24 hours.
- IN-LIFE DATES: From: 04/11/2010 To: 15/11/2010

Positive control substance(s):

yes

Remarks:

25% v/v hexyl cinnamic aldehyde

Vehicle:

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

Concentration:

25 and 50% w/v

No. of animals per dose:

Four female mice per dose

Details on study design:

PRE-SCREEN TESTS:
-Irritation: no evidence
- Systemic toxicity: There were no deaths and no signs of ill health or toxicity were observed during this study. There was no indication of an overt effect of treatment on bodyweight gain.
- Ear thickness measurements: There was no increase in ear thickness which would have been suggestive of irritation.
- Erythema scores: No erythema was observed on the ears of mice from any group on Days 1 to 6.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Results for each treatment group were expressed as the Stimulation Index (SI). This was
derived by dividing the mean DPM/mouse for each treated group and the positive control
group by the mean DPM/mouse in the vehicle control group.
If the SI is 3 or more, the test substance is regarded as a skin sensitizer.
The positive control group is expected to give an SI of 3 or more to demonstrate the validity
of the study.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated at one of three concentrations of the test substance. The
mice were treated by daily application of 25 μL of the appropriate concentration of the test
substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using a automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of four mice received the vehicle alone or the positive control substance in the same manner.

Positive control substance(s):

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

Positive control results:

The SI for the positive control substance hexyl cinnamic aldehyde (HCA) was 11.9 which
demonstrates the validity of this study.

Key result

Parameter:

EC3

Value:

13.6

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The SI (test/control ratios) obtained for 25, 50% w/v and ‘as supplied’ Bis(C9
(branched)alkyl) benzene-1,4-dicarboxylate were 3.3, 6.5 and 11.3 respectively. As a SI of 3
or more was recorded for three of the concentrations tested, Bis(C9 (branched)alkyl)
benzene-1,4-dicarboxylate was considered to have the potential to cause skin sensitization.

EC3 CALCULATION
Based on the results of this study the EC3 value is calculated to be 13.6% w/v

CLINICAL OBSERVATIONS:
There were no deaths and no signs of toxicity were observed during this study.

BODY WEIGHTS
There was no indication of an effect of treatment on bodyweight gain.

 

Group dpm/node and Stimulation Index
Group	Concentration	dpm	Number of lymph nodes per group	dpm/node	Simulation Index †	Result + = positiv - = negativ
1	AOO	2411.85	8.0	301.48	n/a	n/a
2	25% w/v	7966.75	8.0	995.84	3.3	+
3	50% w/v	15630.05	8.0	1953.76	6.5	+
4	As supplied	27280.35	8.0	3410.04	11.3	+
5	HCA 25% v/v	28718.35	8.0	3589.79	11.9	+

† Stimulation Index of 3 or more indicates a positive result

n/a Not applicable

dpm Disintegrations per minute (less background count of 48.65 dpm)

AOO Acetone:olive oil (4:1 v/v) (vehicle control)

HCA Hexyl cinnamic aldehyde (positive control)

Interpretation of results:

study cannot be used for classification

Conclusions:

Bis(C9 (branched)alkyl) benzene-1,4-dicarboxylate is regarded as a potential skin sensitizer.
The EC3 value was calculated to be 13.6% w/v.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Buehler Test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20.11.2012 to 04.04.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Test System: Guinea pig
Species (strain): Cavia porcellus (Hartley)
Animal Source: Mahaveera Enterprises, Hyderabad, India
Number Used: Thirty ( 10 in control and 20 in treatment group) Sex 15 males and 15 females (Females were nulliparous and nonpregnant)
Initial Body Weight (g) on Day 0: Male: Minimum: 370, Maximum: 484; Female: Minimum: 348, Maximum: 436
Age of Animals on Day 0: 10 to 12 weeksTEST ANIMALS
- Source: Mahaveera Enterprises, Hyderabad, India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 to 12 weeks
- Weight at study initiation: Male: 370 - 484 g, Female: 348 -436 g
- Housing: one guinea pig per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19- 23 °C
- Humidity (%): 64-66 %
- Air changes (per hr): Minimum 15 air changes/hr
- Photoperiod (hrs dark / hrs light): 12h light, 12h darkness
- IN-LIFE DATES: From: 17/10/2012 To:26/11/2012

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.2 mL

Day(s)/duration:

6 hours exposure

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.2 mL

Day(s)/duration:

6 hours exposure

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

control group: 5 males/5 females
treatment group: 10 males/10 females

Details on study design:

RANGE FINDING TESTS:
Based on the results of th epilot study, a volume of 0.2 mL 100 % DINT (undiluted) was selected for topical induction applications (days 0, 7, 14) and for challenge exposure on day 28.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Exposure period: 6 h
- Test groups: 1 treatment group ( 10 males, 10 females)
- Control group: 1 control group ( 5 males, 5 females)
- Site: topical to left flank
- Frequency of applications: once
- Duration: The skin reactions were evaluated at 24 h post patch removal on days 1, 8 and 15.
- Concentrations: undiluted

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 28
- Exposure period: 6 h
- Test groups: 1 treatmant group (10 males, 10 females)
- Control group: 1 control group (5 males, 5 females)
- Site: application to the right flank
- Concentrations: undiluted
- Evaluation (hr after challenge): Skin reactions were observed at 24 and 48 h from time of patch removal.

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde

Vehicle:

unchanged (no vehicle)

Concentration:

undiluted

No. of animals per dose:

treatment group: 10 animals/sex
control group: 5 animals/sex

Details on study design:

PRE-SCREEN TESTS:
A patch loaded with 0.2 mL 100% DINT was topically applied to the clipped flanks of two guinea pigs, one male and one female. At the end of a 6h exposure period, the residual test item was removed with cotton soaked in distilled water. The skin reactions were evaluated following the Draize method at 24 and 48 h post patch removal.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Buehler Test
- Criteria used to consider a positive response: Skin reactions were evaluated according to Magnusson and Kligman grading scale.

Positive control substance(s):

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

Positive control results:

The results of the positive control study confirmed the sensitivity of the Guinea Pig Buehler Test Method.

Key result

Reading:

1st reading

Hours after challenge:

30

Group:

test chemical

Dose level:

treatment group (undiluted test material)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

not observed

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

54

Group:

test chemical

Dose level:

treatment group (Undiluted test material)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

not observed

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

In the present Buehler test, a sensitization rate of 0 % at 30 and 54 h follwing challenge exposure, using a non-adjuvant method, was observed.
Since a response of at least 15 % is considreed as postive in non-adjuvant method for the Buehler test, Di-isononyl terephthalate (DINT) is being classified as "Not considered as a positive" according to GHS 2011.

Reference 3

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

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)

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

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.

Positive control results:

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

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.

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.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20.06.13 - 01.12.15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was conducted before the OECD TG 442C was announced. The GLP status was not claimed and no audit of the study report was performed.The Good Laboratory Practice status was not claimed since this study was initiated prior to availabilily of the validated ECVAM DB-ALM protocol.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

The study was conducted before the OECD TG 442C was announced.

Deviations:

not specified

GLP compliance:

no

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The objective of the Direct Peptide Reactivity Assay (DPRA) was to evaluate the reactivity of the t6est ilem, DINT, to synthetic cysteine and lysine peplides for skin sensitizalion assessment. The reactivity of the lest item was evaluated in chemico by monitoring peptide depletion alter 24 hours.

The assay determines the chemical reactivity of the test item to cysteine and lysine peptides, which is a unifying characteristic of most skin sensitizing chemicals (Gerberick et al., 2004, Gerberick et al., 2007). Reactivity (%depletion) is determined following 24 hours contact between lest item and peptide in acetonitrile and detection by liquid chromatography with Ultra-Viole! detection. The protocol and data interpretation are in accordance with the ECVAM protocol used for the validation of the DPRA.

Peptide reactivity was reported as percentage depletion based on the decrease in non-reacled peplide concentration in the sample relative to the average concentration measured in the control.
The amount of non-reacted peptide was calculated from the standard curve for either cysteine or lysine.

Positive control results:

The positive controls samples with Cinnamaldehyde were satisfied. The study was therefore considered to be valid.

Key result

Parameter:

other: peptide reactivity

Remarks:

mean percentage depletion of the cysteine and lysine peptides

Value:

0

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study and considering the solubility issue of test item in the peptide buffer solution, the test item DINT was considered to have no/minimal peptide reactivity and therefore may have no/minimal potential to cause skin sensitization.

Reference 5

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

31.10.14 - 15.11.2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was conducted before the OECD TG 442D was announced. The study is not performed under GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

The study was conducted before the OECD TG 442D was announced.

Deviations:

not specified

GLP compliance:

no

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The Induction of Antioxidant-Response-Element Dependent Gene Activity in the Keratinocyte ARE- Reporter Cell Line KeratinoSens skin sensitization assay is a high-throughput cell-based in vitro test to screen for the skin sensitization potential of chemicals. The KeratinoSens cells (Givaudan, Switzerland) were propagated as a reporter cell line. The KeratinoSens cells are transfected HaCaT keratinocytes that include a 56-base-pair insertion containing the ARE sequence from the AKR1C2 gene, a SV40 promoter, the luciferase gene in the vector pGL4 from Promega, and a stable insertion allowing for cells to be selected in the presence of Geneticin (G418). The signalling pathway with the repressor protein Keap1 and the transcription factor Nrf2, which binds to the antioxidant / electrophile response element (ARE / EpRE), was shown to be a valuable cellular endpoint to detect skin sensitizers in vitro1,2. The induction of luciferase directly indicates the activation of ARE-dependent genes. Additionally, the cytotoxicity of a test article was assessed using cell MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide). Cytotoxicity was determined by measuring the relative conversion of MTT in test article-treated cultures compared to the solvent control.
The experimental design of this study consisted of three definitive assays to determine the maximal induction (Imax), the concentration for maximal gene induction (CImax), the EC1.5 value (concentration for a statistically significant induction of 50% above solvent controls), and a mean IC50 (concentration leading to 50% viability as compared to solvent controls) for the test articles. For each definitive assay, the KeratinoSens cells were cultured in quadruplicate plates for
24 hours, treated with the test articles for 48 hours, and assessed for luciferase induction (3 plates) and cytotoxicity (1 plate).

Positive control results:

The positive control, Cinnamic Aldehyde, show that the test system is valide.

Key result

Parameter:

other: EC 1.5 values (µg/mL)

Value:

400

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

For this test article, and EC1.5 value was obtained in only 1 repetition and, therefore, this test article will not be considered a potential sensitizer as the prediction model requires that 2/3 repetitions produce an EC1.5 value below 200 μg/mL. The EC1.5 value of 1.15 μg/mL, is not averaged with the other 2 repetitions which did not produce an EC1.5 value.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:

The KeratinoSens assay was accepted when the positive control (cinnamic aldehyde) caused an in vitro score that fell within two standard deviations of the historical mean.

Evaluation of Test Results:
A test article was predicted to have sensitization potential if:
1) The EC1.5 value fell below 200 μg/mL in at least 2 of 3 repetitions;
2) At the lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%; and
3) There was apparent overall dose response which was similar between repetitions.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the current prediction model, the test article DINT, Di-iso-nonyl-terephthalate is predicted to be non-sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The endpoint skin sensitisation for the test item Diisononynlterephthalate (DINT) is tested in five in vivo and in vitro tests: Two Local lymph node assays (LLNA) in mice (with and without challenge), one Buehler test in guinea pigs and two in vitro tests (KeratinoSens and DPRA).

The classical LLNA revealed DINT to be a sensitiser based on the calculated EC3 value of 13,6%. Additionally, a modified LLNA with a secondary response (IMDS, Integrated Model for the Differentiation of Chemical-induced Skin Reactions) was performed. The modified LLNA is used to investigate whether the test item can induce specific memory cells or not. The modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes (Homey et al., 1998; Vohr et al., 2000), as also recommended in the update of OECD TG 429. The results for the test with DINT do not point to a specific immunostimulating (skin sensitizing) potential of the test item. It has to be assumed that the positive response in cell proliferation obtained for primary response is exclusively due to irritation or nonspecific cell activation after application of up to 50% DINT. The results show that there is no indication for a skin sensitizing effect after administration of 25 % DINT in this test system as a challenge concentration.

In the Buehler test a sensitisation rate of 0% at 24 and 48 h post patch removal from the time of challenge application was observed using a non-adjuvant method. The two in vitro tests, which were conducted before the current guidelines came into effect also revealed that DINT has no or minimal potential to cause skin sensitisation.

It is well known, that the LLNA gives false positive results for a considerable number of test compounds (Kreiling et al, 2008; Basketter et al., 2009). The occurrence of false positive results is caused by non-specific cell proliferation of lymph node cells as a result of inflammatory processes in the skin (Vohr & Ahr, 2005). The modified LLNA is able to distinguish between a specific (sensitizing) or non-spezific (irritant) stimulating potential of the test item by measuring weights of draining lymph nodes, stimulation indices for cell counts and ear swelling.

Moreover, Kimber & Dearman (2010) present an overview about the impact of phthalates on immune and allergic responses in animals and humans. They conclude that there "is no evidence that phthalates have the potential to directly cause allergic sensitisation, nor is there a reason to suppose that they are intrinsically immunogenic".

Thus, there is evidence that the common LLNA without second response tested with DINT gave a false-positive result. The extended challenge LLNA showed no induction of specific memory cells. The Buehler assay, in which as well a challenge application is applied, comes to the same result that DINT has no skin sensitisation potential.

In a weight of evidence approach, the challenge LLNA is regarded as the most appropriate test to investigate a sensitisation potential of DINT. The results of the modified LLNA are supported by the results of the Buehler and the two in vitro tests, so that it can be concluded that DINT is not regarded as a skin sensitiser.

 

References:

Basketter et al. (2009), Nothing is perfect, not even the local lymph node assay: a commentary and the implications for REACH, Contact Dermatitis, 60: 65-69.

Homey et al. (1998), An integrated Model for the Differentiation of Chemical-Induced Allergic and Irritant Skin Reactions (IMDS). Toxicol. and Appl. Pharmacal., 153, 83-94.

Kimber & Dearman (2010), An assessment of the ability to phthalates to influence immune and allergic responses, Toxicology, 271(3):73-82.

Kreiling et al. (2008), Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT), Food Chem. Toxicol., 46(6):1896-904.

Vohr & Ahr (2005), The local lymph node assay being too sensitive?, Arch. Toxicol., 79(12):721-8.

Vohr et al. (2000), Discrimination Between (Photo )Allergic and (Photo )Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification for skin/respiratory sensitization is indicated according to classification, labeling, and packaging (CLP) regulation (EC) No 1272/2008.