2-(N-methyl-p-toluidino)ethanol

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(N-methyl-p-toluidino)ethanol

EC Number:

220-638-5

EC Name:

2-(N-methyl-p-toluidino)ethanol

Cas Number:

2842-44-6

Molecular formula:

C10H15NO

IUPAC Name:

2-[methyl(4-methylphenyl)amino]ethan-1-ol

Test material form:

liquid: viscous

Specific details on test material used for the study:

The test item is a liquid non-surfactant substance. It was tested directly, without dilution or preparation of a solution.

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 Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour and 10 minutes.
 

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of test item

Duration of treatment / exposure:

10 minutes at 32 ± 1 °C.

Observation period (in vivo):

3,5 hours at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 hours at 32 ± 1 °C

Number of animals or in vitro replicates:

3

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

21.61

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

1.1     Opacity and Permeability Values

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

Table 9.1‑a     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	1059	1044	1022	1032	1013	1009	998	1024	998
(I) Measured values after exposure	1040	1013	1032	1034	1012	1008	282	282	272

Rep. = Replicate

 

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

Table 9.1‑b     Opacity Values Negative Control

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	1.53	2.12	3.01
Opacity after exposure	2.27	3.39	2.60
Opacity Difference	0.75	1.27	-0.41
Mean Opacity Difference	0.54

Rep. = Replicate

 

Table 9.1‑c     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	1.92	4.12	2.97	4.03	2.93	4.03
Opacity after exposure	13.46	17.41	16.17	114.34	114.34	120.00
Opacity Difference	11.54	13.29	13.20	110.31	111.42	115.97
Opacity Difference corrected	11.01	12.76	12.67	109.78	110.88	115.43
Mean Opacity Difference corrected	12.15			112.03

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:

Table 9.1‑d     Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.032
2. Measurement	0.036
3. Measurement	0.033
Mean	0.034

 

Table9.1‑e     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. Measure-ment	0.044	0.043	0.043	0.481	0.479	1.057	1.520	2.009	0.848
2. Measure-ment	0.043	0.039	0.037	0.478	0.482	1.046	1.525	2.025	0.839
3. Measure-ment	0.045	0.039	0.039	0.483	0.475	1.071	1.534	2.061	0.840
1. Measure-ment – blank	0.0103	0.0093	0.0093	0.4473	0.4453	1.0233	1.4863	1.9753	0.8143
2. Measure-ment – blank	0.0093	0.0053	0.0033	0.4443	0.4483	1.0123	1.4913	1.9913	0.8053
3. Measure-ment – blank	0.0113	0.0053	0.0053	0.4493	0.4413	1.0373	1.5003	2.0273	0.8063
Mean of each replicate	0.0103	0.0067	0.0060	0.4470	0.4450	1.0243	1.4927	1.9980	0.8087
Mean of the 3 replicates	0.0077			--			--
Corrected	--	--	--	0.4393	0.4373	1.0167	1.4850	1.9903	0.8010
Corrected mean of the 3 replicates	--			0.6311			1.4254

Rep. = Replicate

 

1.2     IVIS Values

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

Table9.2‑a     IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.90	0.65	134.22%*
	1.37
	- 0.32
Test Item Ethylphthalyl ethyl glycolate	17.60	21.61	25.59%
	19.32
	27.92
Positive Control DMF undiluted	132.05	133.41	5.06%
	140.74
	127.45

 

*Note: the high relative standard deviation of the IVIS of the negative control is due to mathematical reasons, as the respective means are very small.

1.3     Validity

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:

Table9.3‑a     Validity

Parameter	Criterion	Found	Assessment
Mean IVIS of negative control HBSS	≤3	0.65	ok
Mean IVIS of positive control DMF undiluted	53.18 - 138.89	133.41	ok

 

The mean IVIS values for negative and positive controls were within the range of historical data of the test facility (see Annex 2, page22). Therefore, the test system was acceptable.

1.4     Assessment

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no prediction can be made.

Table9.4‑a     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 N-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) showed effects on the cornea of the bovine eye. The calculated mean IVIS was 21.61.

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

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

This in vitro study was performed to assess corneal damage potential of N-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) by quantitative measurements of changes in opacity and permeability in a bovine cornea. The test item N-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. 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. The test item was tested neat. Under the conditions of this test, the test item N-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) showed effects on the cornea of the bovine eye. The calculated mean IVIS was 21.61. According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no prediction can be made. The negative control (HBSS) and the positive control (undiluted dimethylformamide) have met the validity criteria. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Executive summary:

One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old. The test item N-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT) was applied 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 mean IVIS (In Vitro Irritancy Score) was 0.65. 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 mean IVIS was 133.41.

 

Under the conditions of this study, the test itemN-(2-Hydroxyethyl)-N-Methyl-p-Toluidine (MHPT)showed effects on the cornea of the bovine eye. The calculated mean IVIS was 21.61.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP study only. In this case no prediction can be made.