2,4,6-trimethylpyridine

Endpoint summary

Description of key information

The test item was found to be corrosive to skin and in conclusion also severely eye damaging.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-12-19 to 2020-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™, Epi-200-SIT kit and MTT-100 assay, purchased from MatTek Corporation (82105 Bratislava, Slovakia)
- Tissue batch number: Lot No.: 30842

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 1 hour
- Spectrophotometer: microplate reader (Versamax® Molecular Devices)
The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA
- The test item is identified as non-irritant to skin in accordance with UN GHS / EU CLP “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
DPBS (MatTek)
- Amount applied: 30 µL

POSITIVE CONTROL
5% SDS solution in deionised water (MatTek)
- Amount applied: 30 µL

Duration of treatment / exposure:

After pre-warming of the EpiDerm™ tissues was completed, the negative and positive control, and the test item was added atop the tissues. The test item respectively controls were tested in triplicate tissues with an exposure time of 60 minutes. Within this period the 6-well plates were placed in the incubator for 34 minutes at standard incubation conditions. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.

Duration of post-treatment incubation (if applicable):

After treatment and washing, the tissues were incubated in 0.9 mL of fresh assay medium for 24 hours. After this incubation period the medium was changed and the tissues were incubated for another about 18 hours at standard incubation conditions. The complete incubation time was about 42 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 runs with 3 replicates each

Value:

2.18

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the viability compared with the negative control to 2.91%, thus ensuring the validity of the test system
- Acceptance criteria met for variability between replicate measurements: The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according OECD 439: ≤ 18), thus ensuring the validity of the study.

Table 1: Results after treatment with 2,4,6-Collidine and the controls

Treatment Group	Tissue No.	OD 570 nm			Mean OD of 3 Wells	Mean OD of 3 tissues blank corrected	Rel. Viability [%] Tissue 1, 2 + 3	Standard Deviation	Mean Rel. Viability [%]
		Well 1	Well 2	Well 3
Blank		0.039	0.039	0.039	0.039
Negative control	1	1.680	1.603	1.604	1.629	1.656	96.023	3.6	100.0
	2	1.731	1.763	1.746	1.747		103.149
	3	1.711	1.696	1.718	1.708		100.828
Positive control	1	0.089	0.094	0.092	0.091	0.048	3.147	0.2	2.91
	2	0.087	0.087	0.086	0.087		2.857
	3	0.084	0.084	0.085	0.084		2.714
Test item	1	0.080	0.079	0.079	0.079	0.036	2.406	0.2	2.18
	2	0.072	0.072	0.073	0.072		1.999
	3	0.074	0.075	0.075	0.074		2.124

 

Interpretation of results:

other: The test substance is irritant or corrosive to skin. Further information is necessary to decide if it is to be classifed into category 1 or 2 for skin irritation/corrosion.

Conclusions:

In this study and under the experimental conditions reported, the test item is irritant or corrosive to skin according to UN GHS and EU CLP regulation.

Executive summary:

The in vitro study according to OECD Guideline No. 439 was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic colour was not intensive and it did not change colour when mixed with deionised water or isopropanol. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item the mean relative viability value was 2.18% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant or corrosive to skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 February 2020 to 06 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Justification for test system used:

Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional subcategory 1A, optional subcategory 1B and 1C) and non-corrosives. The test protocol may also enable the distinction between severe and less severe skin corrosives.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human EpiDerm model purchased from MatTek
- Tissue batch number: Lot No.: 30850

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
The 6-well plates for the 3 minutes exposure periods were stayed at room temperature in the sterile bench, the 6-well plates for the 60 minutes exposure period were incubated under standard conditions.

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with PBS to remove any residual test material for several times. Excess PBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues have been rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
24-well plates were prepared with 300 μl MTT solution kept under standard conditions until required. Following rinsing, the tissue inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period under standard conditions the tissues were rinsed with PBS and carefully dried with blotting paper.
The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted within about 19.5 h in the refrigerator at 2-8°C without shaking. At the end of the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the inserts were discarded. Subsequently, the plates were shaken for 15 min at room temperature. Additionally, the solution was homogeneous by a pipette before transferred into a 96-well. Per each tissue, 3 × 200 μL aliquots of the formazan solution were transferred in a flat bottom, microtiter plate. 200 μL of isopropanol were added to the wells designated as blanks for 96-well plate.


NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water and isopropanol (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
50 μL (79.4 μL/cm2 according to guideline) of the neat substance were applied onto the tissue surface.

NEGATIVE AND POSITIVE CONTROL
50 μL (79.4 μL/cm2 according to guideline) were dispensed directly onto the tissue surface.

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

For the MTT-Assay, 24-well plates were prepared with 300 μl MTT solution kept under standard conditions until required. Following rinsing, the tissue inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period under standard conditions the tissues were rinsed with PBS and carefully dried with blotting paper.
The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted within about 19.5 h in the refrigerator at 2-8°C without shaking. At the end of the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the inserts were discarded. Subsequently, the plates were shaken for 15 min at room temperature. Additionally, the solution was homogeneous by a pipette before transferred into a 96-well. Per each tissue, 3 × 200 μL aliquots of the formazan solution were transferred in a flat bottom, microtiter plate. 200 μL of isopropanol were added to the wells designated as blanks for 96-well plate.

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure, mean of 2 replicates

Value:

5.56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure, mean of 2 replicates

Value:

64.37

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Colour interference with MTT:
The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water and isopropanol (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (11.55%) and for the 1 hour exposure period (2.71%) thus confirming the validity of the test system and the specific batch of tissue models. After exposure of the tissues to the test item the relative absorbance value was 64.37% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 5.56%. The value after 1 hour did exceed the threshold for corrosivity which is defined to be < 15% after the 1 hour exposure. Therefore, the test item is considered to be corrosive.

Table 1: Results after treatment with 2,4,6-Collidine and the controls after 3 minutes exposure

Treatment Group	Tissue No.	Exposure [min]	OD 570 nm			Mean OD of 3 wells blank corrected	Mean OD of 2 tissues blank corrected	Standard deviation (well 1, 2 and 3)	Mean Rel. Viability [%]	CV [%]
			Well 1	Well 2	Well 3
Blank		3	0.039	0.04	0.040
Negative control	1		2.125	2.04	2.022	2.023	2.065	0.055	100	2.9
	2		2.180	2.123	2.138	2.108		0.030
Positive control	1		0.278	0.264	0.258	0.227	0.239	0.010	11.55	6.9
	2		0.294	0.286	0.288	0.250		0.004
Test item	1		1.436	1.379	1.424	1.374	1.329	0.030	64.37	4.7
	2		1.354	1.315	1.305	1.285		0.026

 

 

Table 2: Results after treatment with 2,4,6-Collidine and the controls after 60 minutes exposure

Treatment Group	Tissue No.	Exposure [min]	OD 570 nm			Mean OD of 3 wells blank corrected	Mean OD of 2 tissues blank corrected	Standard deviation (well 1, 2 and 3)	Mean Rel. Viability [%]	CV [%]
			Well 1	Well 2	Well 3
Blank		60	0.039	0.040	0.040
Negative control	1		2.070	2.027	1.995	1.991	1.937		100	4
	2		1.959	1.904	1.904	1.883
Positive control	1		0.117	0.103	0.099	0.067	0.052		2.71	38.5
	2		0.080	0.077	0.076	0.038
Test item	1		0.149	0.147	0.147	0.108	0.108		5.56	0.6
	2		0.148	0.146	0.147	0.107

 

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The test item is considered to be corrosive based on the results of this study.

Executive summary:

This in vitro study was performed to assess the corrosive potential of 2,4,6-Collidine by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water and isopropanol (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (Dulbecco's Phosphate-Buffered Saline (DPBS)) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value was 64.37% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 5.56%. The value after 1 hour reached the threshold for corrosivity which is defined to be < 15% after the 1 hour exposure. Therefore, the test item is considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item 2,4,6-Collidine is corrosive to skin according to EU CLP and UN GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

9th October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Storage, temperature and transport conditions of ocular tissue:
Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
- Selection and preparation of corneas:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement. At the end of the incubation period, the basal opacity was determined. Only corneae with a value of the basal opacity < 7 were used. Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min +/- 30 sec

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED
Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 10 min exposure


POST-INCUBATION PERIOD:
yes, 2 h

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability:
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was measured with a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
The mean IVIS value of each treated group was calculated from the individual IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS ≤ 3: UN GHS No Category
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55: UN GHS Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

10 min treatment time, mean of three corneas

Value:

52.43

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

With the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.87).
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.87) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
The test item 2,4,6-Collidine was tested undiluted. Relative to the negative control, the test item 2,4,6-Collidine caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 52.43 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation in vitro score	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	0	0.059	0.058	0.89	0.87	0.03	No Category
	0		0.059		0.89
	0		0.059		0.83
Positive Control	82*		0.843*		94.65	98.87	4.26	Category 1
	80*		1.252*		98.79
	87*		1.078*		103.18
2,4,6-Collidine	20*		2.201*		53.02	52.43	1.88	No prediction can be made
	23*		2.063*		53.95
	18*		2.155*		50.33

*corrected values (the mean permeability OD of the negative control is subtracted from the permeability OD of each treated cornea)

Interpretation of results:

study cannot be used for classification

Conclusions:

According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made based on the calculated IVIS score of 52.43.

Executive summary:

The in vitro study according to OECD Guideline 437 was performed to assess the corneal damage potential of 2,4,6-Collidine by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time (t130). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water), neither an increase of opacity nor permeability of the corneae was observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/UN GHS (Cat 1)). Relative to the negative control, the test item 2,4,6-Collidine caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 52.43 according to OECD 437 (see table in chapter 3.8.3).

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (UN GHS) for 2,4,6-Collidine.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

OECD 439

The in vitro study according to OECD Guideline 439 was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic colour was not intensive and it did not change colour when mixed with deionised water or isopropanol. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item the mean relative viability value was 2.18% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant or corrosive to skin. Therefore the study according to OECD Guideline 431 was additionally conducted.

 

OECD 431

The in vitro study according to OECD Guideline 431 was performed to assess the corrosive potential of 2,4,6-Collidine by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water and isopropanol (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (Dulbecco's Phosphate-Buffered Saline (DPBS)) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance value was 64.37% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 5.56%. The value after 1 hour reached the threshold for corrosivity which is defined to be < 15% after the 1 hour exposure. Therefore, the test item is considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item 2,4,6-Collidine is corrosive to skin according to EU CLP and UN GHS.

As a conclusion, the substance is considered to be corrosive to skin.

Eye irritation

The in vitro study according to OECD Guideline 437 was performed to assess the corneal damage potential of 2,4,6-Collidine by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae, the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time. After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water), neither an increase of opacity nor permeability of the corneae was observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/UN GHS (Cat 1)). Relative to the negative control, the test item 2,4,6-Collidine caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 52.43 according to OECD 437 (see table in chapter 3.8.3). In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (UN GHS) for 2,4,6-Collidine.

Based on the skin corrosive property of the test item, the substance was considered to be classified into category 1 (severely eye damaging) for eye irritation/severe eye damage.

 

Justification for classification or non-classification

Based on the results obtained the test item was classified as corrosive to skin (Cat. 1), and as causing irreversible damage to the eyes (Cat. 1) and labelled with H314 (causes severe skin burns and eye damage) according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.