Glycine, N,N'-1,2-ethanediylbis-, reaction products with formaldehyde, iron chloride (FeCl3) and phenol, potassium salts

Endpoint summary

Description of key information

One in vitro skin irritation study was available with several supporting studies of a structurally related chemical. HBED-Fe (UVCB) showed no skin irritation).

One in vitro and one in vivo eye irritation study were available with several supporting studies of a structurally related chemical. HBED-Fe (UVCB) was evaluated as not irritating to the eyes.

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

Study period:

August-October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Details on test system:

Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-029 and 13-EKIN-034 ).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Source: SkinEthic Laboratories, Lyon, France.

Amount/concentration applied:

The solid test substance (10.7-12.2 mg) was applied directly on top of the skin tissue.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min

Value:

109

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

HBED-Fe (UVCB) was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that HBED-Fe (UVCB) did not interact with MTT.
The mean absorption at 570 nm measured after treatment with HBED-Fe and controls are presented below Table 1.
Table 2 shows (between brackets) the mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues was 80%.
The positive control had a mean cell viability of 23% after 15 minutes exposure. The OD570 of two out of three negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated with the substance or positive control was less than 4%. The standard deviation value of the percentage viability of three tissues treated with the negative control was 31%, caused by one out of three tissues. Although the SD was outside the acceptability criteria of the assay, a clear negative result was obtained by the test substance. However, since not all criteria were met, the assay was repeated at request of the sponsor.
In the repeat experiment, the relative mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues was 109% (Table 2). Since the mean relative tissue viability for HBED-Fe (UVCB) was above 50% after 15 minutes treatment HBED-Fe (UVCB) is considered to be non-irritant. The positive control had a mean cell viability of 3% after 15 minutes exposure. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly (Table 1).

Table1            Mean absorption in the in vitro skin irritation test with HBED-Fe (UVCB) - 2nd experiment

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.180	1.134	1.050	1.121	±	0.066
HBED-Fe (UVCB)	1.239	1.252	1.183	1.224	±	0.036
Positive control	0.046	0.042	0.028	0.039	±	0.010

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.045). Isopropanol was used to measure the background absorption.

 

In the first experiment the mean values +/- SD were as follows:

- Negative control: 1.472 +/- 0.454

- HBED-Fe (UVCB): 1.174 +/- 0.085

- Positive control: 0.408 +/- 0.057

Table2            Mean tissue viability in thein vitroskin irritation test with HBED-Fe (UVCB) - 2nd experiment

	Mean tissue viability as % of control
Negative control	100 (100)
HBED-Fe (UVCB)	109 (80)
Positive control	3 (23)

 Between brackets the results of the first experiment are given.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not irritating in an in vitro test (OECD 439).

Executive summary:

This report describes the ability of HBED-Fe (UVCB) to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of HBED-Fe (UVCB) was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch CFC 11140 (F501236001-8) of HBED-Fe (UVCB) were dark red-brown microgranules with a purity of 97.2%. Skin tissue was moistened with 5 μl of Milli-Q water and 10.7 to 12.2 mg of HBED-Fe (UVCB) was applied on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues was 80%. The positive control had a mean cell viability of 23% after 15 minutes exposure. The OD570 (optical density at 570 nm) of two out of three negative control tissues was within the laboratory historical control data range The standard deviation value of the percentage viability of three tissues treated with the substance or positive control was less than 4%. The standard deviation value of the percentage viability of three tissues treated with the negative control was 31%, caused by one out of three tissues. Although the SD was outside the acceptability criteria of the assay, a clear negative result was obtained by the test substance. However, since not all criteria were met, the assay was repeated at request of the sponsor. In the repeat experiment, the relative mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues was 109%. Since the mean relative tissue viability for HBED-Fe (UVCB) was above 50% after 15 minutes treatment HBED-Fe (UVCB) is considered to be non-irritant. The positive control had a mean cell viability of 3% after 15 minutes exposure. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly. Finally, it is concluded that this test is valid and that HBED-Fe (UVCB) is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October-November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species: Albino rabbit, New Zealand White, (SPF-Quality). Recognized by international guidelines as the recommended test system (e.g. EC, OECD)
Source: Charles River France, L’Arbresle Cedex, France
Number of animals: 3 Males
Age and body weight: At start of dosing, the animals were between 12 and 24 weeks old and body weights were at least 1.5 kg.
Identification: Earmark.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were recorded in the raw data and were considered not to have had any effect on the outcome of the study.

Accommodation: Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period

Water: Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures.



IN-LIFE DATES: From: 4. To: 12.July 2007

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Animals were treated by instillation of, on average, 46.1 mg (range 45.7 – 46.3 mg) of the test substance (a volume of approximately 0.1 mL) as the maximum required amount in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.

Duration of treatment / exposure:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 3 weeks later, after considering the degree of eye irritation observed in the first animal.

Observation period (in vivo):

7 days because on day 7 no signs were observed anymore.

Number of animals or in vitro replicates:

3 males

Details on study design:

One hour prior to instillation of the test substance, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.

Five minutes prior to instillation of the test substance, two drops of the topical anesthetic lidocaïne eyedrops (AST Farma BV, Oudewater, The Netherlands) were applied to both eyes.

Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

In order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection.

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effects on cornea

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: no effects on iris

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

1.43

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Instillation of approximately 46 mg of HBED-FE (UVCB) (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the iris and conjunctivae. Iridial irritation grade 1 was observed for all animals at 1 hour after instillation and resolved within 24 hours.
The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in all animals.
No corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Other effects:

Brown staining of the fur on the head and paws, caused by the test substance, was noted during the observation period.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Summary of ocular lesions

 

Anim. No.	Effect	Hours	Days after application					Mean score cornea Days 1/2/3	Mean score iritis Days 1/2/3	Mean score redness Days 1/2/3	Mean score chemosis Days 1/2/3
		1	1	2	3	7	21
551	Cornea Iris Redness Chemosis	0 1 2 2	0 0 2 1	0 0 1 0	0 0 1 0	0 0 0 0	- - - -	0	0	1.3	0.3
575	Cornea Iris Redness Chemosis	0 1 2 2	0 0 2 1	0 0 1 0	0 0 1 0	0 0 0 0	- - - -	0	0	1.3	0.3
576	Cornea Iris Redness Chemosis	0 1 2 2	0 0 2 1	0 0 2 0	0 0 1 0	0 0 0 0	- - - -	0	0	1.7	0.3
Mean all anim.								0.0	0.0	1.43	0.3

 

Interpretation of results:

GHS criteria not met

Conclusions:

Although the test item induced slight irritation, no classification is needed according to OECD-GHS.

Executive summary:

The study was carried out based on the guidelines described in:

OECD No.405 (2012) "Acute Eye Irritation / Corrosion"

EC, No 440/2008, B5: "Acute Toxicity: Eye Irritation/Corrosion"

EPA, OPPTS 870.2400 (1998): "Acute Eye Irritation"

FAMIC Guidelines (2011), including the most recent revisions.

  

Single samples of approximately 46 mg of HBED-FE (UVCB) (a volume of approximately 0.1 mL) were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation.

Instillation of the test substance resulted in effects on the iris and conjunctivae. Iridial irritation grade 1 was observed for all animals at 1 hour after instillation and resolved within 24 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in all animals.

Based on these results, HBED-FE (UVCB) does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

One in vitro skin irritation study was available with several supporting studies of a structurally related chemical. HBED-Fe (UVCB) showed no skin irritation).

One in vitro and one in vivo eye irritation study were available with several supporting studies of a structurally related chemical. The results of the in vitro eye irritation study (slight eye irrititation) did not allow classification. As such an in vivo eye irritation study was also performed. HBED-Fe (UVCB) was evaluated as not irritating to the eyes.

Justification for selection of skin irritation / corrosion endpoint:

One in vitro (GLP) was availabale together with supporting studies with structurally related UVCBs.

Justification for selection of eye irritation endpoint:

One in vitro and one in vivo study (GLP) were availabale together with supporting studies with structurally related UVCBs.

Justification for classification or non-classification

Based on the results of the skin and eye irritation studies the test substance HBED-Fe (UVCB) is not classified and labelled as an eye or skin irritant according to Regulation 1272/2008/EC (CLP).