Bis(2-ethylhexyl) phthalate

Respiratory sensitisation

Administrative data

Endpoint:

respiratory sensitisation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards, described in sufficiently detailled.

Data source

Reference

Reference Type:

publication

Title:

Phthalate treatment does not influence levels of IgE or Th2 cytokines in B6C3F1 mice

Author:

Butala JH, David RM, Gansc G

Year:

2004

Bibliographic source:

Toxicology 201 (2004) 77–85

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

topical application (and challenge) of test substances to mice followed by measurements of serum IgE.
auricular lymph nodes harvesting for measurement of IL-4 and IL-13 proteins and their corresponding mRNAs.
liver weight increase monitoring (for evaluation of skin absorption).

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) phthalate

EC Number:

204-211-0

EC Name:

Bis(2-ethylhexyl) phthalate

Cas Number:

117-81-7

Molecular formula:

C24H38O4

IUPAC Name:

1,2-bis(2-ethylhexyl) benzene-1,2-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): di(2-ethylhexyl)phthalate DEHP
- Physical state: liquid
- Analytical purity: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: no data
- Weight at study initiation: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
"in accordance with regulatory guidelines (NRC, 1996)"

Test system

Route of induction exposure:

dermal

Route of challenge exposure:

other: dermal (on the ears)

Vehicle:

unchanged (no vehicle)

Concentration:

0, 25, 50, 100 %

No. of animals per dose:

positive and negative control groups: 10 females per dose
tests groups:15 females per dose

Details on study design:

A. INDUCTION EXPOSURE
- No. of exposures: 10
- Exposure period: 6 hours / exposure
The application sites for animals given test or positive control substances were covered with fiber pads and semi-occlusive wraps to maintain skin contact and to prevent the mice from licking the treated areas. After 6 hours the wrappings were removed, and the treated areas were cleaned.
- Site: on each flank in 50µl aliquots
- Frequency of applications: once daily
- Duration: 6 hours/day, 5 days/week for 2 weeks
- Concentrations: 0, 25, 50, 100%
- Evaluation: Five mice were taken at the end of the induction period for assessment of liver weights.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 exposure
- Day(s) of challenge: Day 21
Challenge doses of test or control substances were applied in 25 ml aliquots to the ears of the each of the remaining mice except for the untreated control animals that were not treated in either the induction or the challenge phases.
- Site: ear
- Concentrations: 0, 25, 50, 100%
- Evaluation: After an additional week without treatment, the remaining mice were euthanized. Blood samples and auricular lymph nodes were removed for IgE, IL-4, and IL-13 assessments.

Challenge controls:

none

Positive control substance(s):

trimellitic anhydride (TMA)

Negative control substance(s):

2,4-dinitrochlorobenzene (DNCB)

Results and discussion

Results:

All animals survived.
No significant differences in body weight was observed.
Liver weights of DEHP-treated mice were significantly elevated with respect to control.
Phthalate treatment had no effect on IL-4 or IL-13 mRNA levels.

Positive control results:

Serum IgE levels were significantly increased by treatment with the positive control, TMA.
TMA increased IL-4 and IL-13 mRNAs in lymph node cells. The assessment of IL-4 and IL-13 levels in cultured lymph node cells revealed a large and statistically significant increase in cells from mice treated with TMA.

Negative control results:

A small but still significant increase of serum IgE level has been observed in mice treated with DNCB, a contact sensitizer.
IL-4 and IL-13 levels were also elevated in the DNCB-treated groups in most situations but were not significantly different from control values. Neither IL-4 nor IL-13 was elevated in cells from control group mice or mice treated with any of the phthalates.
DNCB treatment increased IL-4 and IL-13 mRNA, but these differences, although statistically significant, were well below levels associated with TMA treatment.
The appropriate responses with the reference compounds demonstrated the specificity of the assay system.

Any other information on results incl. tables

Table 3:  Effect of topically administered DEHP on liver weights in female B6C3F1 mice (n=5).

Dose Group	Body Weight (g)	Liver Weight (g)	% of Body Weight
Vehicle control	16.57 + 1.14	0.816  +  0.079	4.94 + 0.56
DEHP (25%)	16.34 + 0.60	0.984  +  0.044*	6.02 + 0.31*
DEHP (50%)	16.04 + 0.72	1.109  +  0.030*	6.92 + 0.20*
DEHP (100%)	16.70 + 1.16	1.349  +  0.178*	8.05 + 0.56*

* Values significantly different from control (* = P0.05) are indicated by an asterisk.

Table 4:  Total Serum IgE levels in female B6C3F1 mice exposed to DEHP for 14 days.

Administered Material	Total IgE (mg/ml)
Challenge Control	0.69 + 0.19 (10)^1
Vehicle Control	0.74 + 0.08 (10)
TMA (respiratory sensitizer) 25%	6.34 + 1.00** (10)
DNCB (contact sensitizer) 1%	1.76 + 0.32** (10)
DEHP (undiluted)	0.86 + 0.10 (10)
DEHP (50%)	0.89 + 0.12 (10)
DEHP (25 %)	0.60 + 0.05 (10)

  + SE.  The number of animals used in each test is given in parentheses. Values significantly different from vehicle control are noted by an asterisk, ** = p 0.01

Table 5:  IL-4 and IL-13 concentrations from Con A-stimulated, cultured lymph node cells from female B6C3F1 mice exposed to DEHP for 14 days.   

Administered Material	Total IL-4 concentration  (pg/ml)	Total IL-13 concentration (pg/ml)
Assay Controls
Untreated Control	2.73 + 0.48 (5) ^1	5.35 + 1.44 (5)
Vehicle Control	2.73 + 0.48 (5)	3.91 + 0.00 (5)
TMA (respiratory sensitizer) 25%	20.38 + 3.81 **(5)	30.61 + 6.82 **^2 (5)
DNCB (contact sensitizer) 1%	2.73 + 0.48 (5)	5.07 + 1.16 (5)
Test Substance Evaluation
DEHP (undiluted)	2.34 + 0.39 (5)	3.91 + 0.00 (5)
DEHP (50% in vehicle)	2.930+ 0.57 (4) ^3	3.91 + 0.00 (4)^3
DEHP (25 % in vehicle)	2.44 + 0.49 (4) ^3	3.91 + 0.00 (4)^3

Values represent the mean + SE derived from the number of cultures indicated in parentheses. The lower limit of IL-4 detection was 3.91 pg/ml.  Statistical significance is indicated by asterisks (** = p 0.05). 

       

                

Table 6:  IL-4 and IL-13 mRNA concentrations from Con A-stimulated, cultured lymph node cells from female B6C3F1 mice exposed to DEHP for 14 days. 

Administered Material	Total IL-4 mRNA concentration  (pg/ml)	Total IL-13 mRNA concentration (pg/ml)
Untreated Controls
Untreated Control	2.4 + 1.6 (5)^1	4.6 + 2.9 (5)
Vehicle Control	0.0 + 0.0 (5)	2.0 + 1.2 (5)
TMA (respiratory sensitizer) 25%	31.4 + 5.14 (5)	40.8 +  6.1** (5)
DNCB (contact sensitizer) 1%	1.6 + 1.6 (5)	3.4 + 2.9 (5)
Test Substance Evaluation
DEHP (undiluted)	0.0 + 0.0 (5)	0.0 + 0.0 (5)
DEHP (50% in vehicle)	0.0 + 0.0 (4) ^2	1.8 + 1.8 (4)
DEHP (10 % in vehicle)	0.0 + 0.0  (4) ^2	0.0 + 0.0 (4)

¹  Unless otherwise indicated, each culture was prepared from pooled lymph nodes from 2 animals.  Values represent the mean +SE derived from the number of cultures indicated in parentheses.  The lower limit of IL-4 detection was 3.906 pg/ml. 

²  Values represent 3 cultures obtained from pooling of nodes obtained from 2 animals and 1 culture from pooling of nodes obtained from 4 animals.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Conclusions:

Under these experimental conditions, DEHP is not considered as a respiratory sensitizer.

Executive summary:

Di(2-ethylhexyl) phthalate (DEHP) was tested using for respiratory sensitization in B6C3F1 mice. This test involves topical application and challenge of DEHP to mice followed by measurements of serum IgE. 

In addition auricular lymph nodes were harvested for measurement of IL-4 and IL-13 proteins and their corresponding messenger RNAs. Liver weight increase, a measure of peroxisomal proliferation, was monitored to assure that internal dosing had been achieved. 

ELISA and RNAse protection assays demonstrated that DEHP treatment did not significantly affect IgE, IL-4 or IL-13 levels. Similarly IL-4 and IL-13 mRNA levels were not elevated. In contrast, all of these were significantly elevated by trimellitic anhydride (TMA), a respiratory sensitizer used as the positive control in this assay. Liver weights were significantly elevated by DEHP, providing evidence of sufficient percutaneous absorption to induce physiological responses.

Under these experimental conditions, DEHP is not considered as a respiratory sensitizer.