Formaldehyde, reaction products with bisphenol A and diethanolamine, propoxylated

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

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

Test material

Test material information

Constituent 1

Reference substance name:

(Reaction product of 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol, 2-(2-hydroxyethylamino)ethanol and formaldehyde), propoxylated

EC Number:

926-000-9

Cas Number:

1180524-77-9

IUPAC Name:

(Reaction product of 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol, 2-(2-hydroxyethylamino)ethanol and formaldehyde), propoxylated

Test animals / tissue source

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.

Test system

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

Results and discussion

In vitro

Results

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

In vivo

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.