Reaction mass of 2-{[2-(2-hydroxyethoxy)ethoxy]carbonyl}benzoic acid and 2,2'-oxydiethanol

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was condusted in compliance with the principles of GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

(2002)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex: male
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG; Branch Löhndorf; 24601 Löhndorf/Post Wankendorf; Germany
- Age at study initiation: approx. 4-5 months
- Diet and water: ad libitum
- Acclimation period: at least 20 adaption days

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: The surrounding untreated skin served as control

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

Until changes had completely subsided, however for not more than 14 days after application.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: approximately 6 cm² on the dorso-lateral areas of the trunk of each animal
- Type of wrap if used: non-irritant tape

REMOVAL OF TEST SUBSTANCE
- At the end of the exposure time no residual test item had to be removed
- Time after start of exposure: 4 h

SCORING SYSTEM: as specified by Draize

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #1

Time point:

other: 60 minutes

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #2

Time point:

other: 60 minutes

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #2

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #2

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #3

Time point:

other: 60 minutes

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #3

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #3

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Remarks:

(redness and eschar formation)

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 60 minutes

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 60 minutes

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 60 minutes

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #3

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

edema score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Other effects:

The control areas did not show any abnormal findings.
There were no systemic intolerance reactions.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Executive summary:

A well-conducted Acute Dermal Irritation/ Corrosion test (OECD 404) was performed on rabbits with the test substance. An erythema (grade 1) was observed in all animals 60 minutes, in animal nos. 2 and 3 until 48 hours after patch removal. There were no systemic intolerance reactions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study

Principles of method if other than guideline:

The protocol for this study is executed according to the ICCVAM Test Method Evaluation Report: Appendix G: ICCVAM Recommended Protocol for Future Studies Using the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) Test method, November 2006.

GLP compliance:

yes

Species:

other: hen's egg chorioallantoic membrane (HET-CAM)

Details on test animals or tissues and environmental conditions:

The test system used for this study is the fertile Lohmann Brown hen egg (Brinkschulte Josef GmbH & Co.KG, 48308 Senden, Germany), which is incubated for 8 days before test item application.

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control: 0.9 % sodium chloride (300 µl); positive control: 1 % sodium dodecyl sulfate (300 µl)

Amount / concentration applied:

300 µl per egg

Duration of treatment / exposure:

single application

Observation period (in vivo):

300 seconds after substance application

Number of animals or in vitro replicates:

4 eggs per group (negative control, positive control, test substance)

Details on study design:

TEST SYSTEM:
The fertile Lohmann Brown hen eggs (Brinkschulte Josef GmbH & Co.KG, 48308 Senden) were incubated in an incubator with an automatic rotating device (e.g. Ehret GmbH), optimum temperature : 37.5 °C, relative humidity 63 %. After 7 days of incubation, all eggs were candled in order to discard those that were defect and to mark the air bubble. The eggs were replaced into the incubator with the large end upward but not rotated, thus ensuring accessibility to the CAM region. At day 8 of incubation the sections marked for the air bubble were sawed out of the shell. The inner membrane was moistened with NaCI 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing.

METHODS:
This study is used to evaluate the potential ocular irritancy or corrosion of a test item as measured by its ability to induce toxicity in the chorioallantoic membrane of a chicken egg. A 100 % concentration is tested on the chorioallantoic membrane (HET-CAM) of a chicken embryo. Effects are measured by the onset of haemorrhage, vessel lysis or coagulation during the first 300 seconds after application. Times till appearance of each of these endpoints are used to calculate an irritation score. However, there is no clear discrimination between strong irritation and corrosion. The protocol for this study is executed according to the ICCVAM Test Method Evaluation Report: Appendix G: ICCVAM Recommended Protocol for Future Studies Using the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) Test method, November 2006. These tests are also related to OECD 405, with inclusion of the 'Manual of decisions for implementation of the sixth and seventh amendments to Directive 67/548/EEC on dangerous substances (Directives 79/831/EEC and 92/32/EEC).

POSITIVE AND NEGATIVE CONTROL:
Physiological saline solution (0.9 % NaCl, 300 µl) was used as negative control. A SDS (sodium dodecyl sulfate) solution (1 %, 300 µl) was used as positive control (Sigma- Aldrich; Cat.No.: L5750-100G).

APPLICATION OF TEST MATERIAL:
300 µl of the test item was applied to the CAM (4 eggs each). NaCI 0.9 % treated eggs were used as negative contrals, SDS 1 % as positive contral (in
quadruplicate each). Observations of effects to the blood vessels, albumen or embryo over a period of 300 seconds after substance application are determined for each single egg.

OBSERVATIONS:
0= no effect
1 = vasodilatation, slight haemorrhage (H)
2 = vessel lysis, strang haemorrhage (L)
3 = blood-coagulation, albumen -coagulation (C)

The time to the appearance of each of the observations mentioned above has been monitored and recorded. If no effect appeared during the observation period of 300 seconds (observation = 0) the result was assigned as negative for the related endpoint, and the factor set to 0 for this endpoint when calculating the IS.

IRRITATION SCORE:
Following formula was used to generate an irritation score (IS):
IS = 5 x (301-sec H)/300 + 7 x (301- sec L)/300 + 9 x (301- sec C)/300

H= observed start in seconds of haemorrhage reactions
L= observed start in seconds of vessel lysis, strong haemorrhage
C= observed start in seconds of blood-coagulation, albumen-coagulation

Irritation parameter:

other: irritation score (IS)

Run / experiment:

mean

Value:

8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A test substance is considered to cause severe irritation when the IC value is >= 9.

Table 1: Summary of results from HET-CAM test with the test item

 

Compound	Irritation Score (IS)	Assessment
Negative control NaCl 0.9%	0	non irritant
Positive control SDS 1%	9	strong irritant
Test item	8	moderate irritant

Interpretation of results:

other: moderate irritant effect in vitro

Executive summary:

The test item was identified as "moderate irritant" to the chorioallantoic membrane (irritation score (IS) = 8 of maximal 21) under the conditions of the HET-CAM test (Hen's Egg Test - Chorioallantoic Membrane) according to a protocol recommended by ICCVAM, November 2006.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Principles of method if other than guideline:

The HCE model is currently involved in the eye irritation validation conducted by COLIPA following ECVAM guidelines. Furthermore, it is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). This model is recognized as the model of choice and scientifically relevant as documented by several publications (Cotovio et. al., Tox. in Vitro, 24, 2010, 523-537).

GLP compliance:

yes

Species:

other: Assessment of ocular irritation potential of the test substance by determination of cytotoxic effects on a human corneal epithelium (HCE) cell model (exposure 60 min./rt followed by 16 hours incubation at 37 °C, subsequently MTT).

Details on test animals or tissues and environmental conditions:

The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

Vehicle:

unchanged (no vehicle)

Controls:

other: as negative control: Phosphate Buffered Saline (PBS, 30 µL)

Amount / concentration applied:

30 µl per insert

Duration of treatment / exposure:

60 min at room temperature

Observation period (in vivo):

post-exposure incubation: 16 hours (37°C, 5% CO2, maximum humidity)

Details on study design:

The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelia. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item.
Tests were performed in triplets. The test item was applied at a 100% concentration, i.e. 30 µl per insert, for 60 min at room temperature. After the exposure period the inserts were washed carefully with PBS. After a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction was performed. Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item.

Positive control: 1H-1,2,4-Triazole-3-thiol (30 mg, plus 30 µl PBS for moistening)

Irritation parameter:

other: cell viability (%)

Value:

ca. 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.

Summary of the results:

Sample No.	Test item	%Viability
1 - 3	negative control PBS	100.00
4 - 6	positive control 1H-1,2,4 -Triazole-3 -thiol	37.98
7 - 9	test substance	1.64

The test item was detected as irritant under the conditions of this test model.

Interpretation of results:

other: irritant property in vitro

Executive summary:

An in vitro study for assessing ocular irritation of chemicals using a human 3D-corneal epithelial (HCE) cell model was conducted. The cell model, routinely used by the major Cosmetic and Pharmaceutical companies, has been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). The pure test item (30 µl per insert) was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured as about 1.64 % by the MTT conversion assay. A test item is predicted to be an ocular irritant if the mean relative cell viability is ≤ 50 %

after exposure. The cell viability was ca. 2 %, therefore the test item is predicted as ocular irritant under the conditions of this test method.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

An in vitro study for predicting non-specific corrosive properties of the test item was conducted according to OECD TG 431. The undiluted test item (50 µl per insert) was applied topically to a reconstructed human skin model (EST-1000, CellSystems, Germany). After an exposure period of 3 minutes at room temperature and 60 minutes in an incubator (37 °C, 5 % CO2, maximum huminity), the cell viability was 99.54 % and 100.38 %, respectively, as measured by a MTT conversion assay. It is therefore concluded that the test substance is not corrosive to the skin.

The test item was tested in an in vitro skin irritation test on reconstructed human epidermis (EST-1000) according to EU method B.46 (Skin Irritation). The EST-1000 model provides a functional stratum corneum with an underlying layer of living cells. The test item was applied undiluted for 20 min followed by a 42 hour incubation time. Cell viability was determined by the MTT method. The test item was not characterised by a significant impact on cell viability in this in vitro skin irritation study. Cell viability was reduced to ca. 88 % compared to negative control cultures (100%). Thus, the test item is considered to have no skin irritation category as defined in the UN GHS.

In an Acute Dermal Irritation/ Corrosion test (OECD 404) the test item reveals transient, slight irritation (mean erythema score: 0.67) in 2/3 rabbits. There were no systemic intolerance reactions.

Eye irritation:

An in vitro study for assessing ocular irritation of chemicals using a human 3D-corneal epithelial (HCE) cell model was conducted. The cell model, routinely used by the major Cosmetic and Pharmaceutical companies, has been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). The pure test item (30 µl per insert) was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured as about 1.64 % by the MTT conversion assay. A test item is predicted to be an ocular irritant if the mean relative cell viability is ≤ 50 % after exposure. The cell viability was ca. 2 %, therefore the test item is predicted as ocular irritant under the conditions of this test method.

The test item was identified as "moderate irritant" to the chorioallantoic membrane (irritation score (IS) = 8 of maximal 21) under the conditions of the HET-CAM test (Hen's Egg Test - Chorioallantoic Membrane) according to a protocol recommended by ICCVAM, November 2006.

Respiratory Irritation:

No data available.

Justification for selection of skin irritation / corrosion endpoint:
assessment is based on two in vitro studies for the detection of skin corrosives and irritants and an in vivo study

Justification for selection of eye irritation endpoint:
assessment is based on two in vitro studies for the detection of ocular corrosives and severe irritants

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin irritation:

No corrosive and irritant properties were demonstrated in in vitro-assays for the test item. The slight irritation observed in the in vivo study was fully reversible within 72 hours after patch removal.

No classification required according to Regulation (EC) No 1272/2008, Annex I

Eye irritation:

Two validated in vitro test systems provide evidence that the test item has the potential to seriously damage the eyes.

According to Regulation (EC) No 1272/2008, Annex I, the test item shall be allocated to Category 1: (irreversible effects on the eye).

Respiratory irritation:

No data available.