Reaction product of Fatty acids, C18-unstaturated, dimer with 4,7,10-trioxa-1,13-tridecane-diamine

Endpoint summary

Description of key information

Three skin irritation studies and one eye irritation study were conducted on MTDID 18990.

 

Skin Irritation:

 

EpiDerm in vitro system: Irritating (Category 2) when tested according to OECD 439.

 

Irritating (Category 2) when tested according to OECD 404 in rabbits.

 

Irritating (Category 2) when tested in an acute dermal lethality study according to OECD 402.

 

The dermal irritation potential of MTDID 18990 was evaluated in the EpiDerm reconstructed human epidermal tissue model. The study was conducted according to OECD 439. EpiDerm tissues (n=3) were exposed to 30 uL of test article (neat), DPBS (negative control) or SDS (postive control) for 60 minutes at 37C. Following exposure, the tissues were washed 20 times by filling the plate wells with a gentle stream of DPBS and emptying. The plate wells were then filled with fresh media and returned to an incubator at 37C for 24 hours. At 24 hours, the media was replaced with fresh media and incubated for another 18 hours. Following incubation the tissues were patted dry with a tissue and transferred to plates containing 300 uL of MTT solution per well. The tissues were incuabted with MTT for 3 hours. Following incubation they were washed and the wells were filled with MTT extraction soltuion and extracted for 2 hours at room temperature. The OD at 570 nm was measured for each solution and normalized to the mean of the negative control group to calculate % tissue viability. MTDID 18990 -exposed tissues had % viability values of 8, 5, and 19% for a mean tissue viability of 11%. Based on the results of the study (11% mean tissue viability following 60 minute exposure), MTDID 18990 is a skin irritant.

 

The primary skin irritation/corrosion potential of T-6142 (clear brown liquid) was evaluated in New Zealand White rabbits.  This study was performed in compliance with OECD GLP (1981).  The study method was based on 49 CFR 173.137 and 173.136 (1993) and NIH Publication No. 86-23 (1985).  The hair was clipped from the dorsal skin of three rabbits (1 male, 2 females).  The test material (0.5 mL T-6142) was placed under gauze on three clipped sites of each animal.  The torso was wrapped with plastic in an occlusive manner and secured with non-irritating tape for a 3-minute, 1-hour, and 4-hour exposure (1 site/exposure time).  Observations for skin irritation (erythema and edema) were recorded at 30 minutes and at 24, 48, 72, and 96 hours after unwrapping.  The Primary Irritation Index (PII) was calculated.  No evidence of corrosion was observed at any of the test sites for any of the exposure periods.  Slight erythema (scores: 1) and very slight edema (scores: 1) were observed as a result of the 3-minute exposure.  Slight to well-defined erythema (scores: 1-2) and edema (scores: 1-3) were observed as a result of the 1-hour exposure.  As a result of the 4-hour exposure, well-defined to moderate erythema (scores: 2-3) and edema were observed.  Slight irritation was still observed in all animals at Day 7.  The mean individual scores (24-72 hours) for erythema were 2.0, 3.0, and 3.0.  The mean individual scores (24-72 hours) for edema were 1.67, 2, and 2.67.  The PII was 4.78/8.0.  Based on the results of the study (PII = 4.78/8), the test article is a Category 2 skin irritant.

 

The dermal lethality and irritation potential of MTDID 18990 was evaluated in female Wistar rats.  The study was conducted according to OECD 402 in compliance with OECD GLP.  Initially, MTDID 18990 was administered to a single female Wistar Han rat by a single dermal application at 2000 mg/kg body weight for 24 hours in a range finder study. Based on the results, the main study was performed by dosing two females at 2000 mg/kg. All animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15). No mortality occurred. Focal erythema, scales, scabs and/or necrosis was noted at the treated skin of the animals throughout the observation period.  No edema was observed after the exposure.

Animal 1: Erythema scores of 1 were seen 24, 48, and 72 hr after patch removal and lasted until day 7. Mild focal necrosis, mild scales, and mild scabs were observed through day 14. Irritation persisted until study termination.

Animal 2: Erythema (score of 1) was seen at 24 hr but had resolved by 48 or 72 hr. Mild scales were seen on days 3-5. Irritation resolved by day 6.

Animal 3: Erythema scores of 1 were seen 24, 48, and 72 hr after patch removal and lasted until day 13. Mild scales and mild scabs were observed through day 13. Irritation resolved by day 14.

 

These local effects were considered not indicative of systemic toxicity. No clinical signs of systemic toxicity were noted. The body weight gain shown by the surviving animals during the observation period was within the range expected for rats used in this type of study. No abnormalities were found at macroscopic post  mortem examination of the animals.

 

Exposures in this study were approximately 380 mg (rats weighed an average of 191 g), so this is less than the 0.5 g prescribed in the OECD 404 (rabbit) guideline. Exposures were 24 hr, which is much longer than the 4 hr prescribed in the OECD 404 study. However, these results can be used to support classification as a Skin Irritant Category 2 as signs of irritation persisted throughout the study in one animal and until day 13 in a second animal.

 

Eye Irritation:

 

Irritating (Category 2A) when tested according to OECD 437.

 

The objective of this study was to evaluate the eye hazard potential of MTDID 18990 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study was conducted according to OECD 437 in compliance with OECD GLP.  The eye irritancy potential of MTDID 18990 was tested through topical application for 10 minutes. MTDID 18990 was clear to cloudy dark brown-green liquid.  Bovine eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 C. The corneas were incubated for the minimum of 1 hour at 32 C. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group. The test item was applied as it is (750 uL) directly on top of the corneas. The medium from the anterior compartment was removed and 750 ul of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 minutes at 32 C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 minutes at 32  C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group. Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 minutes at 32 C. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 uL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 61 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. MTDID 18990 induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 18 after 10 minutes of treatment.  Based on the results of the study, the test article is a Category 2A eye irritant.

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:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

No deviations ocurred that adversely impacted the integrity of the study.

GLP compliance:

no

Remarks:

This study was conducted prior to the intention to register the substance for REACH. The study was well-documented and is scientifically valid.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot 20125
- Expiration date of the lot/batch: No data
- Purity test date: 21 January, 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, dosed neat

Test system:

human skin model

Source species:

human

Cell type:

other: Human Keratinocytes cultivated in vitro to resemble human epidermis.

Cell source:

other: Proprietary EpiDerm reconstructed human epidermis tissues were purchased from MatTek Corporation

Details on animal used as source of test system:

SOURCE ANIMAL
Proprietary EpiDerm reconstructed human epidermis tissues were purchased from MatTek Corporation

Justification for test system used:

EpiDerm is an appropriate test system per OECD 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Irritation Test (EPI-200-SIT)
- Tissue batch number(s): No data
- Production date: No data
- Shipping date: No data
- Delivery date: 03 February, 2015
- Date of initiation of testing: 02 February, 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 C
- Temperature of post-treatment incubation (if applicable): 37 C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissue inserts were filled and emptied 20 times with fresh DPBS from a wash bottle.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: No data
- Incubation time: 3 hours
- Spectrophotometer: No data
- Wavelength: 570 nm
- Filter: No data
- Filter bandwidth: No data
- Linear OD range of spectrophotometer: No data

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Freezing
- N. of replicates : 2
- Method of calculation used: % Viability of negative test system control

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be an irritant if the mean tissue viability (as a percentage of the negative control) was less than or equal to 50%.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 uL
- Concentration (if solution): Dosed undilluted.

VEHICLE : None

NEGATIVE CONTROL : DPBS
- Amount(s) applied (volume or weight): 30 uL
- Concentration (if solution): No data

POSITIVE CONTROL : SDS
- Amount(s) applied (volume or weight): 30 uL
- Concentration (if solution): 5%

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

19

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Mean % tissue viablity for the test article was 11%.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results of the study (11% mean tissue viability following 60 minute exposure), MTDID 18990 is a skin irritant.

Executive summary:

The dermal irritation potential of MTDID 18990 was evaluated in the EpiDerm reconstructed human epidermal tissue model. The study was conducted according to OECD 439. EpiDerm tissues (n=3) were exposed to 30 uL of test article (neat), DPBS (negative control) or SDS (postive control) for 60 minutes at 37C. Following exposure, the tissues were washed 20 times by filling the plate wells with a gentle stream of DPBS and emptying. The plate wells were then filled with fresh media and returned to an incubator at 37C for 24 hours. At 24 hours, the media was replaced with fresh media and incubated for another 18 hours. Following incubation the tissues were patted dry with a tissue and transferred to plates containing 300 uL of MTT solution per well. The tissues were incuabted with MTT for 3 hours. Following incubation they were washed and the wells were filled with MTT extraction soltuion and extracted for 2 hours at room temperature. The OD at 570 nm was measured for each solution and normalized to the mean of the negative control group to calculate % tissue viability. MTDID 18990 -exposed tissues had % viability values of 8, 5, and 19% for a mean tissue viability of 11%. Based on the results of the study (11% mean tissue viability following 60 minute exposure), MTDID 18990 is a skin irritant.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 402

Version / remarks:

2017

Deviations:

no

Remarks:

No deviations ocurred that negatively impacted the integrity of the study.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Lot 4293484
- Expiration date of the lot/batch: 31 January 2021
- Purity test date: 10 November, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The test article was dosed neat.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : The test article was dosed neat.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 9-12 weeks old
- Weight at study initiation: 184-212
- Housing: On arrival, animals were group housed (up to 5 animals of the same sex together) in polycarbonate cages (Makrolon MIV type; height 18 cm.) and following assignment to the study, animals were individually housed in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room in which the animals were kept was documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 (mean 21)
- Humidity (%): 40-64
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 May, 2019 To: 17 June, 2019

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg body weight

VEHICLE : None, dosed neat.

NEGATIVE CONTROL : An area adjacent to the treated skin were used as negative controls.

POSITIVE CONTROL : None

Duration of treatment / exposure:

24 hours

Observation period:

14 days

Number of animals:

3 females

Details on study design:

TEST SITE
- Area of exposure: 5x7 cm on the back of each rat
- % coverage: Approximately 10%
- Type of wrap if used: The test item was held in contact with the skin with a dressing, consisting of a surgical gauze patch (Surgy 1D), successively covered with Coban elastic bandage. A piece of Micropore tape was additionally used for fixation of the bandages.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, 24 hours after the start of exposure with water.
- Time after start of exposure: 24 hours.

OBSERVATION TIME POINTS : The skin reactions were assessed at approximately 24, 48 and 72 hours after the removal of the dressing and test item.

SCORING SYSTEM:
- Method of calculation: Draize

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: See 'Remarks'

Remarks:

7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

other: See 'Remarks'

Remarks:

Mild (Grade 1 of 4) scabs and necrosis

Basis:

animal #1

Time point:

14 d

Score:

1

Max. score:

1

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Remarks:

Mild (Grade 1 of 4) scabs and necrosis were observed through the end of the study (day 14).

Irritation parameter:

other: See 'Remarks'

Remarks:

Mild (Grade 1 of 4) scales

Basis:

animal #1

Time point:

other: Days 5-10

Score:

1

Max. score:

1

Reversibility:

fully reversible within: See 'Remarks'

Remarks:

10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: See 'Remarks'

Remarks:

48 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

other: See 'Remarks'

Remarks:

Mild (Grade 1 of 4) scales

Basis:

animal #2

Time point:

other: Days 3-5

Score:

1

Max. score:

1

Reversibility:

fully reversible within: See 'Remarks'

Remarks:

5 days

Remarks on result:

positive indication of irritation

Remarks:

Mild (Grade 1 of 4) scales were observed on days 3, 4 and 5.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: See 'Remarks'

Remarks:

Erythema scores of 1 were observed through Day 13, resolving on Day 14.

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

other: See 'Remarks'

Remarks:

Mild (Grade 1 of 4) scabs

Basis:

animal #3

Time point:

other: Days 3-13

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: See 'Remarks'

Remarks:

13 days

Remarks on result:

positive indication of irritation

Remarks:

Mild (Grade 1 of 4) scabs were observed on days 3-13.

Irritation parameter:

other: See 'Remarks'

Remarks:

Mild (Grade 1 of 4) Scales

Basis:

animal #3

Time point:

other: Days 4-9

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: See 'Remarks'

Remarks:

9 Days

Remarks on result:

positive indication of irritation

Remarks:

Mild (Grade 1 of 4) scales were observed on days 4-9.

Irritant / corrosive response data:

No edema was observed after the exposure.
Animal 1: Erythema scores of 1 were seen 24, 48, and 72 hr after patch removal and lasted until day 7. Mild focal necrosis, mild scales, and mild scabs were observed through day 14. Irritation persisted until study termination.
Animal 2: Erythema (score of 1) was seen at 24 hr but had resolved by 48 or 72 hr. Mild scales were seen on days 3-5. Irritation resolved by day 6.
Animal 3: Erythema scores of 1 were seen 24, 48, and 72 hr after patch removal and lasted until day 13. Mild scales and mild scabs were observed through day 13. Irritation resolved by day 14.

Other effects:

- Other adverse systemic effects: No clinical signs of systemic toxicity were observed. Body weights were comparable to rats of this strain and age.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Exposures in this study were ~380 mg (rats weighed an average of 191 g), so this is less than the 0.5 g prescribed in the OECD 404 (rabbit) guideline. Exposures were 24 hr, which is much longer than the 4 hr prescribed in the OECD 404 study. However, these results can be used to support classification as a Skin Irritant Category 2 as signs of irritation persisted throughout the study in one animal and until day 13 in a second animal.

Executive summary:

The dermal lethality and irritation potential of MTDID 18990 was evaluated in female Wistar rats.  The study was conducted according to OECD 402 in compliance with OECD GLP.  Initially, MTDID 18990 was administered to a single female Wistar Han rat by a single dermal application at 2000 mg/kg body weight for 24 hours in a range finder study. Based on the results, the main study was performed by dosing two females at 2000 mg/kg. All animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15). No mortality occurred. Focal erythema, scales, scabs and/or necrosis was noted at the treated skin of the animals throughout the observation period.  No edema was observed after the exposure.

Animal 1: Erythema scores of 1 were seen 24, 48, and 72 hr after patch removal and lasted until day 7. Mild focal necrosis, mild scales, and mild scabs were observed through day 14. Irritation persisted until study termination.

Animal 2: Erythema (score of 1) was seen at 24 hr but had resolved by 48 or 72 hr. Mild scales were seen on days 3-5. Irritation resolved by day 6.

Animal 3: Erythema scores of 1 were seen 24, 48, and 72 hr after patch removal and lasted until day 13. Mild scales and mild scabs were observed through day 13. Irritation resolved by day 14.

These local effects were considered not indicative of systemic toxicity. No clinical signs of systemic toxicity were noted. The body weight gain shown by the surviving animals during the observation period was within the range expected for rats used in this type of study. No abnormalities were found at macroscopic post  mortem examination of the animals.

Exposures in this study were approximately 380 mg (rats weighed an average of 191 g), so this is less than the 0.5 g prescribed in the OECD 404 (rabbit) guideline. Exposures were 24 hr, which is much longer than the 4 hr prescribed in the OECD 404 study. However, these results can be used to support classification as a Skin Irritant Category 2 as signs of irritation persisted throughout the study in one animal and until day 13 in a second animal.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 49 CFR 173.137 and 173.136 (1993)

Version / remarks:

1993

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: NIH Publication Number 86-23 (1985)

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Lot 1456
- Expiration date of the lot/batch: No data
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No data
- Stability under storage conditions: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The test article was dosed neat.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : The test article was dosed neat.
- Treatment of test material prior to testing: None, dosed neat.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HRP Inc, Kalamazoo, Michigan
- Age at study initiation: "Adult"
- Weight at study initiation: 2634-2880 grams
- Housing: Individually in screen-bottom cages.
- Diet (e.g. ad libitum): Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. in a measured amount.
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 30-70
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 January 1995 To: 23 January 1995

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL

VEHICLE : None, applied neat.

NEGATIVE CONTROL : Untreated skin served as a negative control.

POSITIVE CONTROL : None

Duration of treatment / exposure:

Observations were made at 3 minutes, 1 hour and 4 hours after which the test article was removed via a water wash.

Observation period:

3 minutes, 1 hour and 4 hours after the start of the exposure after which the test article was removed via a water wash. Subsequent observations after test article removal were made at 30 minutes, 24, 48, 72 and 96 hours and again at 7 days.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: Dorsal or flank region (1 inch by 1 inch)
- % coverage: No data
- Type of wrap if used: Gauze covered with paper tape and wrapped in Saran wrap.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The test substance was removed via a tap water wash at 4 hours.
- Time after start of exposure: 4 hours.

OBSERVATION TIME POINTS
3 minutes, 1 hour, 4 hours, 24 hours, 48 hours, 72 hours, 96 hours and 7 days.

SCORING SYSTEM:
- Method of calculation: Draize

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: See 'Remarks'

Remarks:

7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: See 'Remarks'

Remarks:

7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: See 'Remarks'

Remarks:

7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

not fully reversible within: See 'Remarks'

Remarks:

7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: See 'Remarks'

Remarks:

7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

not fully reversible within: See 'Remarks'

Remarks:

7 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

As a result of the 4-hour exposure, well-defined to moderate erythema (scores: 2-3) and edema were observed. Slight irritation was still observed in all animals at Day 7. The mean individual scores (24-72 hours) for erythema were 2.0, 3.0, and 3.0. The mean individual scores (24-72 hours) for edema were 1.67, 2, and 2.67. The PII was 4.78/8.0.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results of the study (PII = 4.78/8), the test article is a Category 2 skin irritant.

Executive summary:

The primary skin irritation/corrosion potential of T-6142 (clear brown liquid) was evaluated in New Zealand White rabbits.  This study was performed in compliance with OECD GLP (1981).  The study method was based on 49 CFR 173.137 and 173.136 (1993) and NIH Publication No. 86-23 (1985).  The hair was clipped from the dorsal skin of three rabbits (1 male, 2 females).  The test material (0.5 mL T-6142) was placed under gauze on three clipped sites of each animal.  The torso was wrapped with plastic in an occlusive manner and secured with non-irritating tape for a 3-minute, 1-hour, and 4-hour exposure (1 site/exposure time).  Observations for skin irritation (erythema and edema) were recorded at 30 minutes and at 24, 48, 72, and 96 hours after unwrapping.  The Primary Irritation Index (PII) was calculated.  No evidence of corrosion was observed at any of the test sites for any of the exposure periods.  Slight erythema (scores: 1) and very slight edema (scores: 1) were observed as a result of the 3-minute exposure.  Slight to well-defined erythema (scores: 1-2) and edema (scores: 1-3) were observed as a result of the 1-hour exposure.  As a result of the 4-hour exposure, well-defined to moderate erythema (scores: 2-3) and edema were observed.  Slight irritation was still observed in all animals at Day 7.  The mean individual scores (24-72 hours) for erythema were 2.0, 3.0, and 3.0.  The mean individual scores (24-72 hours) for edema were 1.67, 2, and 2.67.  The PII was 4.78/8.0.  Based on the results of the study (PII = 4.78/8), the test article is a Category 2 skin irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2019

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:

2017

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Lot 4293484
- Expiration date of the lot/batch: 31 January 2021
- Purity test date: 19 December, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light.
- Stability under storage conditions: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: No data
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, tested neat.

FORM AS APPLIED IN THE TEST: The test article was dosed neat.

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): No data
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: No data
- indication of any existing defects or lesions in ocular tissue samples: Eyes with defects were not used for testing.
- Indication of any antibiotics used: No data

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 uL

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 uL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3 corneas per group (Test article, negative and positive control)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 C. The corneas were incubated for the minimum of 1 hour at 32 C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
3 per group (Test article, negative control and positive control)

NEGATIVE CONTROL USED
Yes, physiological saline

SOLVENT CONTROL USED: NA

POSITIVE CONTROL USED :
Yes, ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 uL, 10 minutes

TREATMENT METHOD: Open chamber

POST-INCUBATION PERIOD: Following the 120 minute post-exposure incubation period and opacity measurements, the corneas were incubated in sodium fluorescein solution for 90 minutes at 32 C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

- POST-EXPOSURE INCUBATION: Following the 120 minute post-exposure incubation period and opacity measurements, the corneas were incubated in sodium fluorescein solution for 90 minutes at 32 C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The standard decision criteria were utilized by the contract lab and reported in the final report. Professional judgement was used to assign a Category 2 classification in the event of an IVIS between 3 and 55.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

18

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 1

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 2

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 3

Value:

21

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

12

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein leakage

Remarks:

Permeability Score (Average OD)

Run / experiment:

Mean

Value:

0.389

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: The lab that performed the study is technically proficient in perforing the BCOP. Negative and positive controls performed as expected, indicating that the test system was valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (Ethanol) was 61 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

 Based on the results of the study, the test article is a Category 2A eye irritant.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of MTDID 18990 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study was conducted according to OECD 437 in compliance with OECD GLP.  The eye irritancy potential of MTDID 18990 was tested through topical application for 10 minutes. MTDID 18990 was clear to cloudy dark brown-green liquid.  Bovine eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 C. The corneas were incubated for the minimum of 1 hour at 32 C. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group. The test item was applied as it is (750 uL) directly on top of the corneas. The medium from the anterior compartment was removed and 750 ul of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 minutes at 32 C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 minutes at 32  C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group. Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 minutes at 32 C. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 uL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 61 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. MTDID 18990 induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 18 after 10 minutes of treatment.  Based on the results of the study, the test article is a Category 2A eye irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

A Weight of Evidence (WoE) approach was utilized to assign a Category 2 skin irritation classification to MTDID 18990 based on three studies.

The OECD 402 was conducted on purified MTDID 18990 (97.5% CASRN 68911 -25 -1 and 2.5% CASRN 4246 -51 -9).

The OECD 439 and OECD 404 studies were conducted on a crude MTDID 18990 formulation with approximately 85 -87% CASRN 68911 -25 -1 and higher residual CASRN 4246 -51 -9 at approximately 13 -15%. CASRN 4246 -51 -9 is a Category 1B corrosive and with its presence in the crude mixture at higher concentrations, MTDID 18990 exhibited dermal irritation potential that would warrant classification as a Category 2 irritant in both the OECD 404 and OECD 439 studies. In the OECD 402 study, the purified MTDID 18990 with lower CASRN 4246 -51 -9 residual also exhibited dermal irritation effects that still warrant a Category 2 classification. The overall weight of evidence suggest that purified MTDID 18990 (CASRN 68911 -25 -1) is a Category 2 skin irritant.

Justification for classification or non-classification

Based on the results of the studies, MTDID 18990 is a Category 2 skin irritant and a Category 2A eye irritant.