Dibutyl terephthalate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliable without restriction; study was conducted according to OECD 429 and EU Method B42 guidelines and GLPs.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-Benzenedicarboxylic acid, dibutyl ester

IUPAC Name:

1,4-Benzenedicarboxylic acid, dibutyl ester

Constituent 2

Reference substance name:

Dibutyl terephthalate

EC Number:

217-803-9

EC Name:

Dibutyl terephthalate

Cas Number:

1962-75-0

Molecular formula:

C16H22O4

IUPAC Name:

1,4-dibutyl benzene-1,4-dicarboxylate

Constituent 3

Reference substance name:

Dibutyl benzene-1,4 dicarboxylate

IUPAC Name:

Dibutyl benzene-1,4 dicarboxylate

Constituent 4

Reference substance name:

Terephthalic acid, dibutyl ester; Di-butyl terephthalate; Dibutylterephthalate; DBT

IUPAC Name:

Terephthalic acid, dibutyl ester; Di-butyl terephthalate; Dibutylterephthalate; DBT

Details on test material:

-Test substance: dibutyl terephthalate
-Physical description: colorless liquid
-Batch number: X-29455-010
-Storage conditions: room temperature in the dark
-Date received: 25 October 2004

The integrity of the data relating to identity, purity and stability of the test substance was the responsibility of Eastman Chemical Company and was not provided in the study report.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test animals:
-Strain: CBA/Ca(CBA/CaBkl)
-Source: B&K Universal Ltd, Hull, UK
-Condition of animals: nulliparous and non-pregnant
-Acclimation: 5 days
-Sex: Female
-Body weight (at study start): 15-23 g
-Age: 8-12 weeks
-Identification: unique tail tattoo number
-Method of animal distribution: random selection
-Animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the quality, purpose, or integrity of the study.
-Housing: individually housed in suspended solid-floor polypropylene cages with softwood woodflakes
-Water: local municipal water ad libitum
-Diet: Certified Rat and Mouse Diet (Code 5LF2) (BCM IPS Limited, London, UK) ad libitum

Environmental conditions:
-Temperature: 19-25 °C
-Humidity: 30-70%
-Room lighting: 12:12 light:dark
-Air exchange: 12 air changes per hour

Study Dates:
-Study was performed between 29 November 2004 and 16 December 2004.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25 and 50%; also tested at 100% (i. e., neat, without vehicle)

No. of animals per dose:

5 females per dose

Details on study design:

Details on Study Design:

Concentration, homogeneity and stability of the test substance were not performed since it is not a requirement of the guidelines.

Preliminary screening test:
One mouse was treated by daily application of 25 µL of the neat test substance on the dorsal surface of each ear for three consecutive says. The mouse was observed twice on each day of testing and once a day for three days after the last test substance administration. The body weight was recorded on Day 1 (prior to dosing) and on Day 6. The results suggested that there would be no excessive toxicity or irritation with the test substance at concentrations up to 100%.

Test material administration:
Groups of 5 mice were treated with the test substance at concentrations of 25 and 50% in acetone/olive oil 4:1 (v/v) and at 100% (neat, without vehicle). 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. The test substance was administered using a micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A group of 5 mice received the vehicle alone in the same manner.

3^H-Methyl Thymidine administration:
Five days following the first topical application of the test substance, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3^H-methyl thymidine (3^HTdR:80µCi/mL, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total of 20 µCi to each mouse.

Clinical observations:
All animals were observed twice daily during test substance administration and daily for the next three days. All signs of toxicity or signs of ill health were recorded.

Body weights:
The body weight of each animal was recorded before dosing and prior to termination.

Termination:
Five hours following the administration of 3^HTdR, all mice were killed by carbon dioxide asphyxiation. For each animal of each group the draining auricular lymph nodes were excised and placed in 1 mL of phosphate buffer.

Preparation of single cell suspension:
A single cell suspension of the lymph nodes was prepared by gentle mechanical disaggregation through 200-mesh stainless steel gauze; the lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish. The lymph nodes cell suspension was transferred to a 10 mL centrifuge tube, the petri dish was washed with an additional 5 mL of PBS to remove any remaining lymph node cells, and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 X g) for ten minutes, resuspended in 10 mL of PBS, and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% trichloroacetic acid (TCA).

Determination of 3^HTdR incorporation:
After overnight incubation at 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 X g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3^HTdR incorporation was measured by β-scintillation counting using a Beckman LS6500 scintillation system. The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal (dpm/animal). Results were expressed as the Stimulation Index (SI) obtained by dividing the mean dpm/animal for each treatment group by the mean dpm/animal of the control group.

Statistical analysis:
Group means for the dpm and standard deviations were calculated. Individual and group mean dpm values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets, Dunnett’s Multiple Comparison test was used and for non-homogenous datasets, Dunnett’s T3 Multiple Comparison Method was used.

Interpretation of results:
The test material will normally be regarded as a sensitizer if at least one concentration of the test substance produces a SI of equal to or greater than 3. However, in deciding if the test material was a skin sensitizer, consideration was also given to the dose-response relationship and, where appropriate, statistical significance of the results.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: -A stimulation index of greater than 3 was recorded for the neat test substance. -A stimulation index of less than 3 was recorded for two concentrations of the test substance (25 and 50% v/v).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle: 1227.07 ± 304.76 25%: 2689.63 ± 248.18 (p < 0.01) 50%: 2927.83 ± 492.58 (p < 0.01) 100%: 5870.12 ± 1062.73 (p < 0.001)

Any other information on results incl. tables

Screening Test:

No signs of mortality or systemic toxicity were noted. Body weight was normal.

  

Clinical signs and mortality:

There were no deaths during the study. No signs of systemic toxicity were noted in the test substance or control animals during the study.

  

Body weight:

Body weight changes of the test substance animals during the study were comparable to those of the control group.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

When dibutyl terephthalate was tested in the local lymph node assay, it was determined that dibutyl terephthalate was a sensitizer based on a Stimulation Index score of >3 for the undiluted material.

Based on the positive results for sensitization obtained in the current study, dibutyl terephthalate is classified as a Category 1 sensitizer according to GHS.

Executive summary:

Five CBA/Ca(CBA/CaBkl) female mice per group were exposed to 50 µL (25 µL per ear) dibutyl terephthalate either neat (100%) or at test concentrations of 25% or 50% in a 4:1 acetone:olive oil (v/v) vehicle in the local lymph node assay. Three days after the last test substance administration, the animals were injected via the tail vein with 250 µL of phosphate buffered saline containing 3^H-methyl thymidine and euthanized 5 hours later. Single cell suspensions were made, incubated overnight, and scintillation counts obtained. A stimulation index of greater than 3 was recorded for the undiluted test substance and a stimulation index of less than 3 was recorded for the 25% and 50% concentrations. Under the conditions of the study, it was concluded that dibutyl terephthalate is a sensitizer.