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Description of key information

The available study for skin corrosion/irritation is a validated in-vitro study.
The available study for eye corrosion/irritation is an acceptable non-validated in-vitro study (HET-CAM).
Studies fulfill the validity criteria and are considered reliable.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Validated in-vitro study, GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human epidermis model
Strain:
other: EST 1000
Details on test animals and environmental conditions:
Skin model: Batch no. EST-121112-001; CellSytems® Biotechnology GmbH, 53562 Katharinen, Germany
Amount / concentration applied:
30 µL 1,4-Diethylbenzene were applied to the skin model with a surface area of 0.6 cm2 to uniformly cover the skin surface
Duration of treatment / exposure:
The models were cultivated at 21°C for 20 minutes according to the instructions of the EST1000 supplier CellSystems®. An incubation time with the test item for 20 minutes was recommended by the European Centre for the Validation of Alternative Methods (ECVAM).
Observation period:
Post-treatment incubation period of the rinsed tissues in fresh medium of 42-hours.
Number of animals:
Three replicate tissues were employed
Details on study design:
Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. For the negative control the same dose volume as the test item was used, i.e. 30 µL. The PC item was 5% aqueous sodium dodecyl sulphate (SDS) and was applied to the skin at a volume of 30 µL. The NC item was phosphate buffered saline (PBS)3
Irritation / corrosion parameter:
other: other: % exticntion compared to control
Value:
12.2
Remarks on result:
other:
Remarks:
Basis: mean. Reversibility: no data. Remarks: Test item. (migrated information)
Irritation / corrosion parameter:
other: other: % extinction compared to the control
Value:
0
Remarks on result:
other:
Remarks:
Basis: mean. Reversibility: no data. Remarks: Negative control: phosphate buffered saline. (migrated information)
Irritation / corrosion parameter:
other: other: % extinction compared to the control
Value:
2.3
Remarks on result:
other:
Remarks:
Basis: mean. Reversibility: no data. Remarks: Positive control 5% aqueous sodium dodecy sulphate. (migrated information)
Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.
This Test Guideline used provides an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with UN GHS and EU CLP Category 2
Executive summary:

The cell viabilitywas measured by determining theoptical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.

The test item, 1,4-Diethylbenzene,was applied to the model skin surface. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS)was used as the positive reference item. The mean viability of the cells exposed to the test item was 12.2% of the mean negative controls and, hence, below the 50% cut-off value. The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.The viability of cells treated with the positive reference item, 5% SDS, was 2.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation. All quality criteria required were fulfilled.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, non validated in vitro test, GLP compliant
Qualifier:
according to
Guideline:
other: ICCVAM-Recommended Test Method Protocol: Hen's Egg Test - Chlorioallantoic Membrane (HET-CAM) Test method, published 2010
Deviations:
yes
Remarks:
minor
Principles of method if other than guideline:
The basis of the method is to assess the eye irritancy potential of substances by means of the chorioallantoic membrane of hens' eggs (HET_CAM).
The study was carried out according to : ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) Test Method, published 2010.
This method is increasingly used for the detection of eye mucosa membrane irritants. Eye irritation caused by external contact with chemical substances is characterized by corneal damage and/or conjunctival injury and/or iris defects.
The CAM of fertile eggs incubated for 9 days is a vital vascular membrane with a blood vessel complex. Effects which might produce irritancy in the conjunctiva are assessed by exposing the CAM to liquid or solid test items.



GLP compliance:
yes (incl. certificate)
Species:
other: Chicken eggs
Strain:
other: Fertile white Leghorn chicken eggs
Details on test animals or tissues and environmental conditions:
Weight: 48-51g (according to Study Plan the weight should be 50-60 g)
Age: 7 days
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
300 µL/egg (used undiluted)
Duration of treatment / exposure:
20 seconds
Observation period (in vivo):
300 seconds
Number of animals or in vitro replicates:
3 eggs treated with the test item
Negative control item 3 eggs treated with physiological saline
Positive control items 3 eggs treated with NaOH (0.1 N); 3 eggs treated with SDS (1%)

The solutions were prepared on the day of testing.
Details on study design:
The weight of the eggs ranged from 48 to 60 g and not from 50 to 60 g as stated in the Study Plan.
This minor deviation did not affect the validity and integrity of the scientific results obtained during this study.
Irritation parameter:
overall irritation score
Basis:
mean
Score:
10.1
Remarks on result:
other: 1,4-diethylbenzene
Irritation parameter:
overall irritation score
Basis:
mean
Score:
0
Remarks on result:
other: Negative control 0.9% Na Cl solution
Irritation parameter:
overall irritation score
Basis:
mean
Score:
18.6
Remarks on result:
other: Positive control: 0.1N NaOH
Irritation parameter:
overall irritation score
Basis:
mean
Score:
10.2
Remarks on result:
other: Positive control: 1% aqueous solution sodium dodecil sulphate
Irritant / corrosive response data:
20 seconds after application the CAM was carefully rinsed with 0.9% NaCl solution. The reactions on the CAM were observed over a period of 300 seconds. The time for the appearance of each of the noted endpoints was monitored and recorded, in seconds.
The following endpoints were observed:
- haemorrhage (bleeding from the vessels)
- vascular lysis (blood vessel disintegration)
- coagulation (intra- and extra-vascular protein denaturation)

Whereby,
Haemorrhage time = observed start (in seconds) of haemorrhage reactions on CAM
Lysis time = observed start (in seconds) of vessel lysis on CAM
Coagulation time = observed start (in seconds) of coagulation formation on CAM.

The evaluation consists in to giving scores of each to the three HET-CAM points at the three points 0.5, 2 and 5 minutes.

The numerical time-dependent scores for lysis, haemorrage and coagulation are summed to give a single numercial value indicating the potential of the test substance on a scale with a maximum value of 21. The mean value of three eggs facilitates an assessment by a classification scheme analogous to the Draize categories.

Table 1. Scoring scheme for irritation testing

 Effect  score (0.5min)  score (2min)  score (5min)
 lysis  5  3  1
 haemorrage  7  5  3
 coagulation  9  7  5

The observed effects were grades for each egg by an irritation score (IS) according to a formula

Table 2: IS Classificatio Scheme

 IS score range  Irritation Assessment
 0 -0.9  Practically none
 1 -4.9  Slight
 5 -8.9  Moderate
 9 -21  Strong
Interpretation of results:
highly irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions 1,4-diethylbenzene was a strong irritant in an experiment employing the chorioallantoic membrane of hens' eggs (HET-CAM) as model.
Executive summary:

The purpose of this study was to determine the eye irritancy potential of 1,4-Diethylbenzene by means of the chorioallantoic membrane of hens' eggs (HET-CAM). Eye irritation caused by external contact with chemical substances is characterized by corneal damage and/or conjunctival injury and/or iris defects.

The of fertile eggs incubated for 9 days is a vital vascular membrane with a blood vessel complex.

Three eggs each were treated with 300 µL1,4-Diethylbenzene/egg and with the control items. 0.9% NaCl solution was used as the negative control item. 0.1 NSodium hydroxide (NaOH)and 1% aqueous sodium dodecyl sulphate (SDS) were used as the positive control items. The administration volume for the control items was 300 µL per egg.

After administration of the test item blood vessels including the capillary system and the albumen were examined and scored for irritant effects (haemorrhage, coagulation and lysis) during 5 minutes.

The eggs treated with1,4-Diethylbenzene revealed an effect with an irritation score (IS) of 10.0. The test item was considered to be a strong irritant.

The positive control items 0.1or 1% SDS caused the expected effect with irritation scores (IS) of 18.6 or 10.0, respectively and, hence, were well within the historical data-range. No effects were observed in the negative control 0.9% NaCl solution. Hence,the HET-CAM assay is considered to be valid.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation: the test method used OECD 439, is an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with REACH Regulation. The result is considered sufficient for classification of 1,4 -diethylbenzene as Skin Irritant Category 2 according to CLP Regulation and as Xi; R38 according to the criteria of DSD Directive. This has been done following the Integrated Testing Strategy as described in ECHA Guidance R7a. Therefore no further testing on skin irritation is proposed.

Eye Irritation: the test method used is an in-vitro acceptable non validated (HET-CAM) method, but the results are considered conclusive for classification as Eye Damage Cat.1 according to the criteria of Regulation EU 1272/2008 and as Xi;R41 according to the criteria of DSD Directive. This has been done following the Integrated Testing Strategy as described in ECHA Guidance R7a. Therefore no further testing on eye irritation is proposed.


Justification for selection of skin irritation / corrosion endpoint:
GLP study Klimisch score 1. Only one study available.

Justification for selection of eye irritation endpoint:
GLP study Klimisch score 1. Only one study available.

Effects on skin irritation/corrosion: moderately irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Skin irritation: 1,4 -diethylbenzene is classified as Skin Irritant Category 2 according to the CLP Regulation, and Xi; R38 Irritating to skin according to Directive 67/548/EEC.

Eye irritation: the test performed shows that 1,4 -diethylbenzene is highly irritating. Following the guidances (Figure R.7.2 -3) to assess for Classification it is concluded that 1,4 -diethylbenzene has to be classified as Eye Damage Category 1 based on the results of the in vitro test. According to Annex 1 to Directive 67/548/EEC, 1,4 -diethylbenzene is classified as a Xi;R41 Risk of serious damage to eyes.