2,2-dimethylthiazolidine

Endpoint summary

Description of key information

- skin irritation: Category 1 (corrosive) based on GHS criteria; OECD 404 (12 May 1981); RL1; GLP; mean scores: erythema 4/4/4 and edema 2/-/-. In two animals oedema could not be scored due to eschar/crust formation. The irritation was not reversible within 21 days after exposure.

 

- eye irritation: Category 1 (irreversible effects on the eye) based on GHS criteria, OECD 437 (2013); RL 1, GLP, calculated mean in vitro irritancy scores: negative control: 3.80; positive control: 216.40; test substance: 92.40; cut-off criteria for Cat 1: > 55

No further in vitro test was performed because the substance is directly, chemically reacting with MTT and no reliable read out is possible in MTT reduction based tests.

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

Study period:

1989-06-14 to 1989-08-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: Stable under storage conditions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was applied undiluted as delivered by the sponsor

Species:

rabbit

Strain:

New Zealand White

Remarks:

SPF Quality

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, West-Germany
- Age at study initiation: approx. 13 weeks
- Weight at study initiation: 2.805 - 3.257 kg
- Housing: Individually in cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least five days under test conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30-85%
- Air changes (per hr): 7.5-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1989-06-21 To: 1989-07-12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: adjacent untreated skin area

Amount / concentration applied:

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

Duration of treatment / exposure:

4 hours

Observation period:

22 days

Number of animals:

3 females

Details on study design:

TEST SITE
- Approximately 24 hours before treatment, the dorsal fur was shaved with an electric clipper, exposing an area of approximately 100 square centimeters (10 cm x 10 cm).
- Area of exposure: 2x3 cm (surgical gauze patch loaded with test substance)
- Type of wrap if used: surgical gauze patch mounted on an Micropore tape (3M, St. Paul, U.S.A.). The dressing was wrapped around the abdomen and secured with an elastic bandage (Coban, 3M, st. Paul, U.S.A.).

REMOVAL OF TEST SUBSTANCE
- Washing: remaining test substance was removed using a dry tissue and subsequently a tissue moistened with tapwater.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
The skin reactions were assessed at approximately 60 minutes, 24, 48 and 72 hours and at 7, 14 and 21 days after the removal of the dressings and test article.

SCORING SYSTEM:
- according to Draize, J.H., Woodard, G. and Calvery, H.O., 1944
- Method of calculation: in the study report the PII was calcluated by combining the average skin irritation scores for erythema and oedema after 24 and 72 hours

Note: Where the scoring procedure and the clinical judgement were not in agreement, the assessment of irritancy was based on the latter.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal: #2, #3

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Remarks:

severe effects

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal: #2, #3

Time point:

24/48/72 h

Reversibility:

not reversible

Remarks on result:

not determinable

Remarks:

Oedema could not be scored due to eschar formation/crust.

Irritant / corrosive response data:

refer to table with individual findings.

Other effects:

- Other adverse local effects:
A possible corrosive effect was evident on the skin in two animals.
In animal #2 and #3 brown / grey discolouration of the treated skin area at the 60 minutes observation. Subsequent eschar and crust formation.
In the area of application no persistent staining of the treated skin by the test article was observed.

- Other adverse systemic effects:
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

2,2-Dimethylthiazolidine: Irritant/corrosive response data for each animal at each observation time up to removal of each animal from the test

Score at time point / Reversibility	Erythema	Edema
	Max. score: 4	Max. score: 4
60 min	4/4/4	3/-^a/-^a
24 h	4/4/4	2/-^a/-^a
48 h	4/4/4	2/-^a/-^a
72 h	4/4/4	2/-^a/-^a
7 d	4/4/4	3/-^a/-^a
14 d	0/4/4	0/-^a/-^a
21 d	0/4/4	0/-^a/-^a
Average 24h, 48h, 72h	4/4/4	2/-^a/-^a
Reversibility*)	c/n/n.	c/n/ n.
Average time (unit) for reversion	n.	n.

Note:

a = Oedema could not be scored due to eschar formation/crust.

*) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

 

Judgment is based on erythema and edema calculated as mean scores for each animal following grading at 24, 48 and 72 hours after removal of the test material, in consideration of reversibility.

The following mean scores were calculated: erythema  4/4/4 and edema  2/-a/-a. In two animals oedema could not be scored due to eschar/crust formation.

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The test material 2,2-dimethyl thiozolidine was severely irritating in the in vivo skin irritation test under the experimental conditions described in this report. A possible corrosive effect was evident on the skin in two animals.
The following 24/48/72 h mean scores were calculated: erythema 4/4/4 and edema 2/-/-. In two animals oedema could not be scored due to eschar/crust formation. The irritation was not reversible within 21 days after exposure in two animals.

Executive summary:

In a primary dermal irritation study, performed according to OECD guideline 404, three Albino rabbits were dermally patch exposed for 4 hours to 0.5 mL of the test substance 2,2-dimethylthiazolidine. Test sites were covered with semi-occlusive dressing. Animals were then observed for consecutive 21 days.

Irritation was scored by the method of DRAIZE. The following 24/48/72 h mean scores were calculated: erythema 4/4/4 and edema 2/-/-. In two animals oedema could not be scored due to eschar/crust formation. The irritation was not reversible within 21 days after exposure.

In this study, 2,2-dimethylthiazolidine is considered to be corrosive to skin according to the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008). Because data is not sufficient for sub-categorisation the substance is classified in Category 1 and has to be labeled with H 314.

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:

key study

Study period:

2017-06-13 to 2017-08-17

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator, covered with nitrogen

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport in 1 L containers with 500 mL HSS and 1 % penicillin / streptomycin solution, transport of the containers in coolers on ice.
- On the day of experiment the containers with the eyes were transferred in an incubator at 32 °C (± 1 °C) for about 2 hours.

- Time interval prior to initiating testing: 1 day

- Indication of any existing defects or lesions in ocular tissue samples:
Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.

- Indication of any antibiotics used: 1% penicillin / streptomycin solution

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): 0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours at 32°C (± 1 °C)

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.

On the next day (day of experiment) the containers with the eyes were transferred in an incubator at 32 °C (± 1 °C) for about 2 hours.

For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin I streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour in the incubator at 32 °C (± 1 °C).

QUALITY CHECK OF THE ISOLATED CORNEAS
After approximately 1 hour, the MEM medium was aspirated and the chambers were filled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean± 1 standard deviation were selected for the actual test and assigned to the test groups.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
isotonic saline solution (0.9 % NaCI)

POSITIVE CONTROL USED
1 % NaOH in isotonic saline solution (w/v), clear solution

APPLICATION DOSE AND EXPOSURE TIME
750 µL of negative and positive control and of the test item.
Exposure time 10 minutes at 32 °C (± 1 °C)

TREATMENT METHOD:
Closed chamber: the holes of both chambers were sealed with adhesive tape.

POST-INCUBATION PERIOD: 3 hours in total

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removaL During the final rinse cycle the corneas were rinsed again with clear MEM medium in order to remove residues of the dye. The anterior chamber was filled with the MEM medium again to avoid drying out of the cornea.
- POST-EXPOSURE INCUBATION: approx. 2 hours at 32 °C (± 1 °C)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort V3.4 SP6 software from Testo AG, Lenzkirch). The validation of the opacitometer was carried out under the study number T80820 17.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490); ELISA- Reader (Bio-Tek EL 808, Software Gen5).


SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)

DECISION CRITERIA: the decision criteria as indicated in the OECD guideline 437 was used: IVIS > 55 severely irritating to the eye/corrosive (Category 1)

Irritation parameter:

in vitro irritation score

Value:

92.4

Negative controls validity:

valid

Remarks:

IVIS = 3.8

Positive controls validity:

valid

Remarks:

IVIS = 216.4

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

BCOP Results after 10 min incubation:

Test group		Opacity	Permeability at 490 nm	In vitro Irritation Score	Mean In vitro Irritation Score
Negative control	1	0.6	0.006	0.7	3.8 (SD 3.9)
	2	8.1	0.006	8.1
	3	2.6	0.006	2.7
Positive Control	4	195.3	0.652	205.1	216.4 (SD 10.5)
	5	216.7	0.613	225.9
	6	205.8	0.829	218.2
Test item	7	80.9	0.832	93.4	92.4 (SD 12.0)
	8	63.1	1.122	79.9
	9	75.7	1.870	103.8

Summary of results

	Mean opacity value	Mean permeability value	In vitro irritancy score (IVIS)
Negative control isotonic saline solution	3.7	0.006	3.8
Positive control 1% NaOH	206.0	0.698	216.4
Test item 2,2-Dimethylthiazolidine	73.2	1.275	92.4

 

Interpretation of results:

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

Conclusions:

In this BCOP assay, 2,2-Dimethylthiazolidine caused an increase of the corneal opacity and permeability. The calculated mean in vitro score was 92.40. This is far above the threshold of > 55, which is indicative for a response inducing serious eye damage. Classification in GHS Category 1 is required.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of 2,2-dimethylthiazolidine (98.9 % a.i.) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 July 2013 and EU method B.47, December 2010.

The corneae were incubated with the test substance and controls for 10 min. After rinsing with saline, the corneae were incubated for another 2 h at 32 ± 1°C. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS > 55 is defined as a corrosive or severe irritant.

The test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro score was 92.40.

The positive control (1% NaOH) increased the opacity and permeability of the corneae (mean in vitro score 216.40) corresponding to a classification serious eye damage Category 1. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 3.80).

 

Since the mean in vitro irritancy score of the test substance was > 55, 2,2-dimethylthiazolidine is considered to be severely irritating/ corrosive (Cat 1) in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on available and relevant data, the test substance needs to be classified for skin irritation/corrosion according to regulation (EC) 1272/2008 into Category 1 and should be labeled with H 314: "Causes severe skin burns and eye damage".

Corrosion is confirmed in an in-vitro eye irritation study.

Based on available and relevant data, the test substance needs to be classified for eye irritation according to regulation (EC) 1272/2008 into Category 1. Labelling with H 318"Causes serious eye damage" is not indicated because of redundancy.