m-toluic acid

Endpoint summary

Description of key information

Acute dermal irritation/corrosion: not irritating (equivalent or similar to OECD 404)

Eye damage/irritation: corrosive/severe irritant to the eyes (OECD 437, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

pH not evaluated, observation after 30-60 min not conducted; Exposure conditions were more intesive than required by the OECD guideline 404 , in respect of duration and coverage. Each animal was treated at clipped intact as well as abraded skin sites.

Principles of method if other than guideline:

Exposure conditions were more intensive than required by OECD Guideline 404 such that appliction duration was 24 hours hours instead of 4 hours under, type of coverage was occlusive instead of semiocclusive and each of 6 animals was treated at clipped intact as well as abraded skin sites.

GLP compliance:

not specified

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: pulverisation with mortar, moistened with distilled water

Species:

rabbit

Strain:

other: Kbs:JW

Remarks:

japanese white rabbit

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kitayama Labes Co., Ltd.
- Weight at study initiation: 2.1 - 2.5 kg
- Diet (ad libitum): solid feed RC-4 (obtained from Oriental Yeast K.K)
- Water ( ad libitum): tap water
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C
- Humidity (%): 55 ± 6 %

Type of coverage:

occlusive

Preparation of test site:

other: clipped, partly left intact, partly abraded (stratum corneum was removed with injection needles)

Vehicle:

other: distilled water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g per application site, clipped intact as well as abraded skin areas were treated at each animal.

Duration of treatment / exposure:

24 hours

Observation period:

7 days

Number of animals:

6 male animals

Details on study design:

TEST SITE
- Area of exposure: within a circle of 3 cm (according to 7 cm²)
- Type of wrap if used: two layers of gauze patch fixed in a closed patch with Tegaderm (3M) and Ju Picot 5 (obtained from Tokyo Material Research Institute)

REMOVAL OF TEST SUBSTANCE
- removal with gauze soaked in water

OBSERVATION TIME POINTS
- daily after application period. Reported values for 24, 48, 72 and 168 hours

SCORING SYSTEM:
- Method of calculation: scoring of erythema and edema was conducted according to Draize method after 24, 48, 72 and 168 hours.
(Please refer to the field 'Any other information on materials and methods incl. tables')

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: intact skin; 24 hour occlusive exposure

Other effects:

no effects observed

Table 1: Results

Items	Erythema		Oedema
Observation time	24	72	24	72
Rabbit No.
1 int	0	0	0	0
Abr	0	0	0	0
2 int	0	0	0	0
Abr	0	0	0	0
3 int	0	0	0	0
Abr	1	0	0	0
4 int	1	0	0	0
Abr	1	0	0	0
5 int	0	0	0	0
Abr	0	0	0	0
6 int	0	0	0	0
abr	0	0	0	0

Int: intact skin

Abr: abraded skin

1 out of 6 rabbits displayed at the intact site and 2 out of 6 rabbits displayed at the abraded skin sites mild erythema (Draize score 1) after 24 hours, which was reversible after 48 hours.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is non-irritating to the skin.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-23

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:

2013-07-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry; < 30 °C

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Characteristics of donor animals: cattle was between 19 and 34 months old.
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.

Vehicle:

physiological saline

Remarks:

0.9 % NaCl

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of the test substance mixture
- Concentration (if solution): 20 % w/v concentration
The test item was mixed with the vehicle to give a concentration of 20 % w/v using ultrasonic technique. The sonicated suspension was incubated at 32 °C for 1 hour. Prior to application, the mixture was re-suspended by vortexing and administered directly.

Duration of treatment / exposure:

4 hours ± 5 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not required

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with pre-warmed RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
- an initial measurement was performed on each of the corneas using the opacitometer.
- three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas.
- the illuminance of each cornea was read and recorded.
- only corneas that had an initial illuminance reading I > I0/1.1651 lux (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 μL of the test substance mixture or the control substance was introduced into the anterior chamber (closed-chamber method).
- after 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed at least three times with MEM (containing phenol red).
- once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete RPMI and an illuminance measurement was performed.
- each cornea was observed visually and pertinent observations were recorded.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation were recorded.
- after the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete RPMI.
- 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.
- then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated annually. This I0 value is than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance could be calculated and corneas below this value were discarded.
- change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490).
- any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.
- final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
For the IVIS cut-off values for identifying test substances as inducing serious eye damage and test substances not including eye irritation or serious eye damage please refer to table 1 in the field "Any other information onmaterial and methods incl. tables" below.

ACCEPTABILITY CRITERIA
- the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Value:

58.66

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All 3 corneas treated with m-toluic acid showed slight opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: the in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The evaluation of acceptability criteria was performed by using the following historical data:
- for evaluation of the validity of the positive control, the historical mean IVIS score as obtained from 2015 until 2017 was used (please refer to the tables in the field "Any other information on results incl. tables").
- for the evaluation of the validity of the negative control, the historical upper limits of the opacity and permeability values as obtained in 2017 were used (please refer to the tables in the field "Any other information on results incl. tables" below)

Please also refer for results to the field "Any other information on results incl. tables" below

Table 1: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	1.98	2.96	0.98
2		2.09	6.01	3.92
3		2.16	3.78	1.62
MV		2.08	4.25	2.17
4	Positive Control	3.50	112.05	108.5	106.38
5		3.54	83.03	79.49	77.32
6		3.74	87.36	83.62	81.45
MV		3.59	94.15	90.55	88.38
7	Test Item	2.31	49.11	46.80	44.63
8		0.97	59.42	58.45	56.28
9		1.69	73.82	72.13	69.96
MV		1.66	60.78	59.13	56.95

MV = mean value

Table 2: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.019
2		0.022
3		0.028
MV		0.023
4	Positive Control	1.473	1.450
5		2.465	2.442
6		3.065	3.042
MV		2.334	2.311
7	Test Item	0.091	0.068
8		0.092	0.069
9		0.228	0.205
MV		0.137	0.114

MV = mean value

Table 3: In vitro irritation score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.98	0.019
2		3.92	0.022
3		1.62	0.028
MV		2.17	0.023	2.52
4	Positive Control	106.38	0.624
5		77.32	1.182
6		81.45	2.222
MV		88.38	1.343	123.05
7	Test Item	44.63	0.068
8		56.28	0.069
9		69.96	0.205
MV		56.95	0.114	58.66

MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until August 2017

	IVIS Positive Control – Imidazole 20%
Mean Value (MV)	125.12
Standard Deviation (SD)	17.75
MV-2xSD	89.63
MV+2xSD	160.62
Number of Replicates providing Historical Mean: 28

Positive controls are updated after every single experiment or at least every 3 months

Table 5: Historical data on opacity and permeability of the positive control (Imidazole 20 %) from August 2017 until October 2017

Incubation; 240 min
Number of Replicates providing Historical Mean	Cornea No.	Opacity		Permeability		IVIS
		Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
1	4	122.785	121.861	0.662	0.624
	5	117.173	116.249	1.220	1.182	133.42
	6	102.655	101.731	2.260	2.222
2	4	108.553	106.381	1.473	1.450
	5	79.491	77.319	2.465	2.442	123.05
	6	83.618	81.446	3.065	3.042
3	4	55.644	56.308	2.200	2.209
	5	71.511	72.175	1.348	1.337	92.64
	6	65.148	65.812	2.040	2.029
Mean Value (MV)		89.620	88.809	1.859	1.837	116.370
Standard Deviation (SD)		23.998	23.370	0.740	0.745	21.195
MV-2xSD		41.624	42.069	0.379	0.347	73.980
MV+2xSD		137.615	135.549	3.340	3.328	158.760

Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until August 2017

	IVIS Negative Control – NaCl 0.9 %
Mean Value (MV)	1.23
Standard Deviation (SD)	0.77
MV-2xSD	-0.31
MV+2xSD	2.78
Number of Replicates providing Historical Mean: 28

Negative controls are updated after every single experiment or at least every 3 months

Table 7: Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until October 2017

Number of Replicates providing Historical Mean	Cornea No.	Opacity	Permeability	IVIS
		Change of Opacity Value	OD490 Value
1	1	0.234	0.008	1.49
	2	1.738	0.008
	3	0.800	0.098
2	1	0.978	0.019	2.52
	2	3.920	0.022
	3	1.617	0.028
3	1	-0.149	0.009	-0.50
	2	-0.415	0.015
	3	-1.427	0.009
1	1	-0.57	0.009	-0.27
	2	-0.11	0.013
	3	-0.53	0.004
2	1	-0.25	0.004	0.66
	2	0.80	0.005
	3	1.17	0.008
Mean Value (MV)		0.520	0.017	0.780
Standard Deviation (SD)		1.296	0.023	1.254
MV-2xSD		-2.072	-0.029	-1.727
MV+2xSD		3.112	0.064	3.287

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to the bovine corneal opacity and permeability assay, since the IVIS was > 55 (IVIS score: 58.66), the test item m-toluic acid can be considered as requiring classification for eye irritation or serious eye damage.
According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is classified for serious eye damage (Category 1; H318).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

m-Toluic acid did not show a skin irritation potential in an in vivo acute dermal irritation/corrosion test equivalent to OECD test method 404. According to the criteria of REGULATION (EC) No 1272/2008 and its subsequent amendments and corrections, m-toluic acid does not have to be classified and has no obligatory labelling requirement for skin irritation.

m-Toluic acid induced a mean in vitro irritation score (IVIS) of 58.66 in a bovine corneal opacity and permeability assay according to OECD Guideline 437, which indicates on a serious eye damaging potential of the substance. Therefore, the substance is classified as Category 1 (H318: Causes serious eye damage) in accordance with Regulation (EC) No 1272/2008 and its subsequent amendments and corrections. m-Toluic acid is not classified for respiratory irritation (STOT SE category 3) because of lacking data.