Formaldehyde, reaction products with bisphenol A and diethanolamine, propoxylated

Endpoint summary

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:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU Method B.46 (Skin Irritation)

GLP compliance:

yes

Species:

other: in vitro reconstructed human epidermis EST-1000 (Cell Systems, St. Katharinen, Germany)

Strain:

other: strain: not applicable

Details on test animals or test system and environmental conditions:

The model used for this study has a functional statum corneum with an underlying layer of living cells. The barrier function of the stratum corneum is adequate, as has been shown by the supplier.

Type of coverage:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control: physiological saline solution (0.9% NaCl); Positive control: sodium dodecyl sulfate (5% SDS)

Amount / concentration applied:

30 µl test substance per insert (three replicates)

Duration of treatment / exposure:

20 min

Observation period:

42 hours post-treatment incubation period

Number of animals:

not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure period 20 min., followed by a 42 hours post-treatment incubation

Value:

17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

The test item was characterised by a significant impact on cell viability. Thus, the test item should be regarded as skin irritant according to UN GHS category 2.

Summary of results:

Sample No.	Test item	% Viability
1 -3	Negative Control (0.9% NaCl)	100.00
4 -6	Positive Control (0.5% SDS)	1.86
7 -9	M 530	17.06

Interpretation of results:

Category 2 (irritant)

Remarks:

Migrated information

Executive summary:

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 characterised by a significant impact on cell viability in this in vitro skin irritation study. Cell viability was reduced to ca. 17 % compared to negative control cultures (100%). Thus, the test item should be regarded as skin irritant according to UN GHS category 2.

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline 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 mg 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:

8d incubation of chicken eggs

Value:

6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

The test item was identified as "moderate irritant" to the chorioallantoic membrane (irritation score (IS) = 6) under the conditions of this assay.

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%	10	strong irritant
Test item	6	moderate irritant

Interpretation of results:

other: positive

Executive summary:

The test item was identified as "moderate irritant" to the chorioallantoic membrane (irritation score (IS) = 6 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.

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 100.25 % and 101.16 %, 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 characterised by a significant impact on cell viability in this in vitro skin irritation study. Cell viability was reduced to ca. 17 % compared to negative control cultures (100%). Thus, the test item should be regarded as skin irritant according to UN GHS category 2.

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 0.4 % 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. 1 %, 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) = 6 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.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin irritation:

No corrosive but irritant properties were demonstrated in in vitro-assays for the test item.

According to Regulation (EC) No 1272/2008, Annex I, the test item shall be allocated to Category 2: Irritant.

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.