Reaction products of neodecanoic acid and titanium (IV) isopropoxide

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

Study period:

The study was performed between 02 September 2009 and 07 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Draft Test Guideline (version 4)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspectection: 19-08-2009 Date of Signature: 04-03-2009

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Not applicable

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

no

Amount / concentration applied:

TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
10µl of of the test material was applied to the epidermis surface.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used

Duration of treatment / exposure:

15 Minutes & 42 hour post exposure incubation

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure:
10µl of the test material was applied to the epidermis surface.

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.

- Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant (I) R38

Mean tissue viability is >50% : Non-Irritant (NI)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15-minute exposure

Value:

36.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was 36.8% after a 15-minute exposure.

Other effects:

No

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared blue/white which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤20%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was ≥0.6 and the SD value of the percentage viability was ≤20%. The negative control acceptance criterion was therefore satisfied.

Table1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540of tissues	Mean OD540of triplicate tissues	±SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Material	0.966	0.916	0.05	105.5	100*
	0.912			99.6
	0.870			95.0
Positive Control Material	0.085	0.073	0.01	9.3	8.0
	0.078			8.5
	0.057			6.2
Test Material	0.341	0.337	0.02	37.2	36.8
	0.351			38.3
	0.320			34.9

SD=    Standard Deviation

*=     The mean viability of the negative control tissues is set at 100%

 

Table2 : Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material	-	-	-
Positive Control Material	++	++	++
Test Material	+	+	+

MTT visual scoring scheme
-          =         blue tissue (viable)
+         =         blue/white tissue (semi-viable)
++       =         tissue is completely white (dead

Interpretation of results:

irritating

Remarks:

Migrated information EXAMPLE Criteria used for interpretation of results: expert judgment

Conclusions:

The test material was considered to be an Irritant.

Executive summary:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Methods:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200µl samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 36.8% after a 15 -minute exposure.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:  The test material was considered to be an Irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed on 13 November 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

no guideline followed

Guideline:

other: The rabbit enucleated eye test is used (in-house), as a first stage in the assessment of ocular irritancy potential.

Deviations:

no

Principles of method if other than guideline:

The study was designed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye.
The rabbit enucleated eye test is used (in-house), as a first stage in the assessment of ocular irritancy potential. The preferred species of choice is the rabbit. The assay has undergone inter-laboratory validation and has been shown to reliably detect test materials that are negligible, or moderate to severe ocular irritants.
The strain of rabbit used in these laboratories has been shown to produce satisfactory responses using known ocular-irritants and non-ocular irritants during in-house validation (see attached Appendix 3). The results of the study are believed to be of value in predicting the ocular irritancy potential of the test material in man.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1% w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope (Keeler Ltd, Windsor, Berks; UK). Corneal thickness values were also recorded using the DGH-1000 Ultrasonic pachymeter (DGH Technology Inc, Solana Beach, CA). Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes.

Vehicle:

unchanged (no vehicle)

Remarks:

For the purpose of the study the test material was used as supplied.

Controls:

other: Two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride).

Amount / concentration applied:

The test material was used undiluted as supplied. A volume of 0.1 ml of the test material was applied as evenly as possible to the surface of the cornea.

Duration of treatment / exposure:

After ten seconds the test material was washed off the cornea using a minimum of 20 ml of saline solution (approximately 32°C).

Observation period (in vivo):

Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.

The thickness of the cornea was measured using an ultrasonic pachymeter. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.

The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment.

The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment.

Number of animals or in vitro replicates:

Three eyes were treated with test material, two additional eyes remained untreated for control purposes.

Details on study design:

Test Material Administration
Three eyes were treated with test material, two additional eyes remained untreated for control purposes. The treatment eye was removed from the superfusion apparatus whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally into a petri dish.
The test material was used undiluted as supplied. A volume of 0.1 ml of the test material was applied as evenly as possible to the surface of the cornea. After ten seconds the test material was washed off the cornea using a minimum of 20 ml of saline solution (approximately 32°C).

Observations
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment, according to the numerical evaluation given in Appendix 2 adopted from Advances in Modern Toxicology: Dermatoxicology, 4th Ed, (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749-815.
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment, according to the numerical evaluation given in attached Appendix 2. This was carried out using the cobalt blue filter of the slit lamp biomicroscope, following application of Fluorescein Sodium drops.

Irritation parameter:

cornea opacity score

Remarks:

Corneal Opacity

Basis:

other: Maximum score

Time point:

other: Maximum score

Max. score:

1

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Maximum score for Corneal Opacity 1

Irritation parameter:

cornea opacity score

Remarks:

Area of corneal opacity

Basis:

other: Maximum score

Time point:

other: Maximum score

Max. score:

3

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Maximum score for Area of corneal opacity 3

Irritation parameter:

cornea opacity score

Remarks:

Corneal Swelling (%)

Basis:

other: Mean score

Time point:

other: 60 Minutes

Score:

13.8

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Mean score @ 60 Minutes 13.8 %

Irritation parameter:

cornea opacity score

Remarks:

Corneal Swelling (%)

Basis:

other: Mean score

Time point:

other: 120 Minutes

Score:

35.3

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Mean score @ 120 Minutes 35.3 % which meets or exceeds cut-off value indicating a severe ocular irritant

Irritation parameter:

cornea opacity score

Remarks:

Corneal Swelling (%)

Basis:

other: Mean score

Time point:

other: 240 Minutes

Score:

58

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Mean score @ 240 Minutes 58.0 % which meets or exceeds cut-off value indicating a severe ocular irritant

Irritation parameter:

cornea opacity score

Remarks:

Condition of Corneal Epithelium

Basis:

other: Maximum score

Time point:

other: Maximum score

Reversibility:

not specified

Remarks on result:

other: Sloughing was seen which meets or exceeds cut-off value indicating a severe ocular irritant

Irritant / corrosive response data:

Corneal Opacity
Individual scores for corneal opacity are given in Table 1.
Some loss of transparency was noted in all test eyes. No corneal effects were noted in the control eyes during the study period.

Corneal Thickness and Condition
Individual and mean corneal thickness measurements and corneal swelling calculations are given in Table 2 and Table 3. The condition of the corneal epithelium following treatment is given in Table 4.
Corneal swelling of the test eyes during the study period was considerably greater than to that observed in the control eyes over the same period.
Sloughing of the corneal epithelium was noted in two test eyes. The condition of the corneal epithelium of one test eye and the control eyes appeared normal during the study period.

Fluorescein Uptake
Individual scores for fluorescein uptake are given in Table 5.
Fluorescein uptake was noted in the test eyes 240 minutes following test material application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Interpretation of Results

The data for all endpoints was assessed and an estimate of the test material ocular irritancy potential was made based on the following cut-off values:

REET Parameter*	REET Cut-Off Value
Maximum Corneal Opacity (Corneal Cloudiness x Area)	> or = 4
Maximum Fluorescein Uptake (Intensity x Area)	> or = 4
Mean Corneal Swelling (mins): 60, 120, 240	> or = 25%
Corneal Epithelium Observations	Any with pitting, mottling or sloughing

Endpoints included corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling). For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 minutes) was calculated based upon the pre‑treatment value as follows:

(mean corneal thickness post – treatment) – (mean corneal thickness post equilibration) x 100

Mean corneal thickness post equilibration

A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 minutes post treatment observation periods.

A negative ocular irritancy potential may require further investigation using an in vivo ocular irritation study.

*= Any parameter that meets or exceeds the cut-off values indicates a severe eye irritant.

Table1              Individual Scores for Corneal Opacity

	Test Eyes												Control Eyes
Chamber Number	1A				3A				5A				2A				4A
Time After Treatment (minutes)	60	120	180	240	60	120	180	240	60	120	180	240	60	120	180	240	60	120	180	240
Degree of Corneal Opacity	0	0	0	1	0	1	1	1	0	1	1	1	0	0	0	0	0	0	0	0
Area of Corneal Opacity	0	0	0	1	0	1	1	1	0	2	3	3	0	0	0	0	0	0	0	0

Table2              Individual Measurements for Corneal Thickness (µm)

Test Eyes
Time After Treatment (minutes)		60						120						180						240
Corneal Position		oc	1	2	3	4	mean	oc	1	2	3	4	mean	oc	1	2	3	4	mean	oc	1	2	3	4	mean
Chamber Number	1A	408	450	397	386	398	407.8	434	468	443	425	460	446.0	435	521	443	457	497	470.6	460	630	453	457	523	504.6
	3A	426	423	401	420	422	418.4	441	587	425	551	529	506.6	455	657	431	592	612	549.4	463	677	444	682	693	581.0
	5A	439	437	441	437	443	439.4	502	455	575	621	631	556.8	736	459	640	705	703	648.6	721	471	699	756	753	680.0
Control Eyes
Time After Treatment (minutes)		60						120						180						240
Corneal Position		oc	1	2	3	4	mean	oc	1	2	3	4	mean	oc	1	2	3	4	mean	oc	1	2	3	4	mean
Chamber Number	2A	389	353	362	383	362	369.8	386	337	365	373	359	364.0	369	343	345	360	352	353.8	363	329	343	355	354	348.8
	4A	421	374	373	404	398	394.0	411	376	372	403	405	393.4	401	367	372	390	384	382.8	400	369	355	383	371	375.6

oc=    Optical centre

Table3              Determination of Corneal Swelling (%)

Test Eyes
Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a
1A	Post equilibration	347.8	N/A	3A	Post equilibration	368.0	N/A	5A	Post equilibration	397.4	N/A
	60 Post treatment	407.8	17.3		60 Post treatment	418.4	13.7		60 Post treatment	439.4	10.6
	120 Post treatment	446.0	28.2		120 Post treatment	506.6	37.7		120 Post treatment	556.8	40.1
	180 Post treatment	470.6	35.3		180 Post treatment	549.4	49.3		180 Post treatment	648.6	63.2
	240 Post treatment	504.6	45.1		240 Post treatment	581.0	57.9		240 Post treatment	680.0	71.1

 

Control Eyes
Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Test Eyes
								Mean corneal swelling 1 hour following treatment 13.8% Mean corneal swelling 2 hours following treatment 35.3%+ Mean corneal swelling 4 hours following treatment 58.0%+
2A	Post equilibration	353.2	N/A	4A	Post equilibration	384.4	N/A
	60 Post treatment	369.8	4.7		60 Post treatment	394.0	2.5	Control Eyes
	120 Post treatment	364.0	3.1		120 Post treatment	393.4	2.3	Mean corneal swelling 1 hour following treatment 3.6% Mean corneal swelling 2 hours following treatment 3.8% Mean corneal swelling 4 hours following treatment 0.0%
	180 Post treatment	353.8	0.2		180 Post treatment	382.8	0.0
	240 Post treatment	348.8	0.0		240 Post treatment	375.6	0.0

a=     % Corneal swelling =

N/A=  Not applicable

+ =    Meets or exceeds cut-off value and therefore indicates a severe eye irritant

Table4              Corneal Epithelium Condition

Test Eyes
Chamber Number	Time After Treatment (minutes)
	60	120	180	240
1A	Normal	Normal	Normal	Normal
3A+	Normal	Sloughing	Sloughing	Sloughing
5A+	Normal	Sloughing	Sloughing	Sloughing

 

Control Eyes
Chamber Number	Time After Treatment (minutes)
	60	120	180	240
2A	Normal	Normal	Normal	Normal
4A	Normal	Normal	Normal	Normal

+ = Meets or exceeds cut-off value indicating a severe ocular irritant

Table5              Individual Scores for Fluorescein Uptake (240 Minutes Post Dosing)

	Test Eyes			Control Eyes
Chamber Number	1A	3A	5A	2A	4A
Intensity of Fluorescein Uptake	1	1	2	0	0
Area of Fluorescein Uptake	2	2	3	0	0

Interpretation of results:

highly irritating

Remarks:

Migrated information Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo. Criteria used for interpretation of results: expert judgment

Conclusions:

Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.

Executive summary:

Introduction. 

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man. 

Methods. 

0.1 ml of the test material was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32°C ± 1.5°C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride).

Results.

Maximal ocular irritation observations recorded for the test eyes were as follows:

Corneal Opacity		Fluorescein Uptake		Corneal Swelling (%)						Condition of Corneal Epithelium
				Test Eyes^a			Control Eyes^b
Cldy	Area	Int	Area	60 mins	120 mins	240 mins	60 mins	120 mins	240 mins
1	3	2	3	13.8	35.3+	58.0+	3.6	2.7	0.0	Sloughing+

Conclusion. 

Following assessment of the data for all endpoints the test material was considered to have the potential to cause severe ocular irritancy in vivo.

a=      For each time point the swelling recorded is the mean of three eyes

b=      For each time point the swelling recorded is the mean of two eyes

Cldy=  Corneal cloudiness

Int=     Intensity of fluorescein uptake

Mins= Minutes following treatment

+=      Meets or exceeds cut-off value indicating a severe ocular irritant

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test substance was found to be non-corrosive to skin in the OECD 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis) Test. In the OECD 439 (In Vitro Skin Irritation: reconstructed Human Epidermis) Test skin irritation effects were noted for the test substance. The very slight erythema observed in rats up to 4-5 days in the acute dermal toxicity study indicates that there is potential for skin irritation and the material is classified and labelled accordingly (CLP Category 2).

 

The ocular irritation potential of the test substance was tested in a rabbit enucleated eye test (REET) in place of an in vivo eye irritation test because the test substance was suspected to be strongly irritating and/or corrosive. Treatment of enucleated rabbit eyes with the undiluted test substance for 10 seconds resulted in increased corneal swelling, damage to the corneal epithelium and corneal opacity. Assessment of the data for all endpoints indicated the notified chemical has the potential to cause severe ocular irritancy in vivo. Accordingly, it is classified and labelled as causing serious eye damage (CLP Category 1).

Justification for selection of skin irritation / corrosion endpoint:
Only one study available

Justification for selection of eye irritation endpoint:
Only one study available

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

The test data support classification of the registered substance as a skin irritant (Category 2) and severe eye irritant (Category 1) according to the criteria laid down in Regulation (EC) No 1272/2008 (i.e. CLP).