Terpene Dimers

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25. Apr. 2018 to 03. May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

In experiment 1 the permeability and the IVIS of the test item was not calculated, because the experiment was discontinued before the second opacity measurement. This can be seen as uncritical, because the experiment was declared invalid and repeated. The deviation was assessed and signed by the study director on 15. May 2018.

Deviations:

yes

Remarks:

see remarks.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Terpene Dimers

EC Number:

948-032-2

IUPAC Name:

Terpene Dimers

Test material form:

liquid

Details on test material:

Batch no. DF1710011

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primigenius Taurus (fresh bovine corneas)

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt So-lution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour 30 minutes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µl

Number of animals or in vitro replicates:

3

Details on study design:

After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, test item and positive control were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:
 
Closed Chamber Method
The respective substance (negative control solution, test item or positive control) was ap-plied by pipetting 750 µL of the appropriate liquid through the refill hole in the holder on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded.

Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the closed chamber method, a sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

meam of the three experiments

Value:

2.52

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

5.59

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

1.51

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

0.47

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The mean IVIS of the negative control has to show an IVIS ≤ 3.
The validity criteria and findings are given in the following table: Validity
Parameter Criterion Found Assessment
IVIS of negative control
HBSS ≤ 3 1.01 ok
IVIS of positive control
DMF undiluted 36.44 – 149.99 100.81 ok

Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Any other information on results incl. tables

Opacity and Permeability Values

The illuminance (unit: LUX) values which were measured before and after exposure are given in the following table:

Illuminance Values

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	997	1030	1013	969	1011	1024	1047	965	1034
(I) Measured values after exposure	983	1007	1010	853	998	992	348	334	362

Rep. = Replicate

 

The values in the following tables present the calculated opacity values, according to evaluation (see chapter 8.1):

Opacity Values Negative Control

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.56	2.19	2.89
Opacity after exposure	4.18	3.14	3.01
Opacity Difference	0.61	0.95	0.13
Mean Opacity Difference	0.56

Rep. = Replicate

 

Opacity Values Test Item and Positive Control

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	4.81	2.97	2.43	1.51	4.99	2.03
Opacity after exposure	10.82	3.52	3.78	83.73	88.89	78.96
Opacity Difference	6.01	0.55	1.35	82.21	83.90	76.94
Opacity Difference corrected	5.45	-0.01	0.79	81.65	83.33	76.37
Mean Opacity Difference corrected	2.08			80.45

Rep. = Replicate

 

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.038
2. Measurement	0.037
3. Measurement	0.034
Mean	0.036

 

Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1. Measurement	0.058	0.090	0.051	0.076	0.167	0.045	1.468	0.976	1.825
2. Measurement	0.059	0.089	0.051	0.076	0.166	0.044	1.474	0.968	1.822
3. Measurement	0.055	0.091	0.051	0.074	0.170	0.046	1.482	0.965	1.827
1. Measurement – blank	0.0217	0.0537	0.0147	0.0397	0.1307	0.0087	1.4317	0.9397	1.7887
2. Measurement – blank	0.0227	0.0527	0.0147	0.0397	0.1297	0.0077	1.4377	0.9317	1.7857
3. Measurement – blank	0.0187	0.0547	0.0147	0.0377	0.1337	0.0097	1.4457	0.9287	1.7907
Mean of each replicate	0.0210	0.0537	0.0147	0.0390	0.1313	0.0087	1.4383	0.9333	1.7883
Mean of the 3 replicates	0.0298			--			--
Corrected	--	--	--	0.0092	0.1016	-0.0211	1.4086	0.9036	1.7586
Corrected mean of the 3 replicates	--			0.0299			1.3569

Rep. = Replicate

 

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.93	1.01	70.18%
	1.76
	0.35
Test Item Terpene Dimers	5.59	2.52	107.1%
	1.51
	0.47
Positive Control DMF undiluted	102.78	100.81	3.37%
	96.89
	102.75

 

Note: the high relative standard deviations of the IVIS of test item and negative control are due to mathematical reasons, as the respective means are very small.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
Classification Scheme
IVIS UN GHS
≤ 3 No category
> 3 and ≤ 55 No prediction can be made
> 55 Eye damage Category I
In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category I. The test item Terpene Dimers showed no effects on the cornea of the bovine eye. The calculated IVIS (In Vitro Irritancy Score) is 2.52.

The experiment is considered as sufficient for the classification of the test item, because two of the three replicates of the test item lead to the same assessment for the test item.

Executive summary:

Two experiments were performed, but the first experiment was aborted, because of a technical problem at the opacitometer. The raw data are kept in the GLP-archive of the test facility but are not reported. The second experiment performed was valid.

 

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item Terpene Dimers was brought onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) is 1.01.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 100.81.

 

Under the conditions of this study, the test item Terpene Dimers showed no effects on the cornea of the bovine eye. The calculated IVIS is 2.52.

 

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.