1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Endpoint summary

Description of key information

- Skin irritation: not irritating, New-Zealand White rabbit, OECD TG 404, Zelenak 2010

- Eye irritation: not irritating, male, New-Zealand White rabbit, OECD TG 405, Tavazsi 2010

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:

29 Sep 2010 to 20 Oct 2010

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:

April 24, 2002

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number of animals per test: 3 (males)
- Age at treatment: ~17 weeks
- Acclimatization: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.
- Housing: Rabbits were individually housed in AAALC approved metal wire rabbit cages (65x65 cm with height of 45 cm). Cages are of an open wire structure and cages are placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Diet: ad libitum.
- Water: The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system. The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 17.1-21.4°C
- Relative humidity: 40 -87%
- Ventilation: 15-20 air exchanges/hour.
- Lighting periods: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: 29 Sep 2010 to 20 Oct 2010

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g per animal

Duration of treatment / exposure:

4 hours

Observation period:

3 weeks

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm back and the flanks of the animal
- Type of wrap if used: Adhesive hypoallergenic plaster; the entire trunk of the animals was wrapped with plastic wrap held in place with an elastic stocking

REMOVAL OF TEST SUBSTANCE
- Washing: The dressing was removed and the skin was flushed with lukewarm tap water to clean the application site
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
- 1, 24, 48, 72 hours, 1, 2 and 3 weeks after the end of exposure.

SCORING SYSTEM:
- Scoring system: The skin reaction was assessed according to the numerical scoring system listed in the Commission Directive 2004/73/EC, April 29, 2004 which was based on the Draize scoring system.
- Method of calculation: The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. An animal was positive when the mean score was 2 or greater. The test was positive for irritation when at least 2 animals were positive for the same endpoint (erythema/eschar or oedema).

OBSERVATIONS:
- Viability/mortality, clinical signs and dermal findings were recorded.
- Body weights were recorded at the beginning and at the end of experiment.

Irritation parameter:

erythema score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 3 weeks

Irritation parameter:

edema score

Basis:

animal: #1 and #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

overall irritation score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

1.22

Max. score:

8

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

At observations 1 hour after patch removal, very slight oedema (score 1) was observed in 2 animals. Oedema persisted in 1 animal and was observed at the 24- and 48 hour observations. Very slight erythema (score 1) was recorded in all animals from 1 hour after patch removal and at all subsequent observations up to and including the 1 week observations. At the 2 week observations very slight erythema (score 1) was still observed in 2 animals. Dry cracked skin surface was observed in all animals at the 1 week observation and in 1 animal at the 2 week observation.
As signs of irritation were observed after 2 weeks, irreversibility could not be determined and the study was therefore extended by 1 week. All animals fully recovered and showed no local signs at the 3 weeks observation.

Table 1. Skin Irritation Scores - Individual Values

Sex	Evaluation Interval*	Erythema	Oedema	Cumulative
				Score	Mean
male		1	1	2.00
male	1 hour	1	0	1.00	1.67
male		1	1	2.00
male		1	0	1.00
male	24 hours	1	0	1.00	1.33
male		1	1	2.00
male		1	0	1.00
male	48 hours	1	0	1.00	1.33
male		1	1	2.00
male		1	0	1.00
male	72 hours	1	0	1.00	1.00
male		1	0	1.00
male		1	0	1.00
male	1 week	1	0	1.00	1.00
male		1	0	1.00
male		1	0	1.00
male	2 weeks	1	0	1.00	0.67
male		0	0	0.00
male		0	0	0.00
male	3 weeks	0	0	0.00	0.00
male		0	0	0.00

* Examinations were performed at the specified times after removal of the dressing.

Table 2. Skin Irritaion Scores – Mean Values After 24, 48 and 72 hours

Animal number	Sex	Erythema	N	Oedema	N	Primary Skin Irritaion Index
1	male	1	N	0	3	1.22
2	male	1	N	0	3
3	male	1	N	0.66	3
Mean score		1		0.22

N – number of available datapoints

Clinical observations

No clinical signs of systemic toxicity were observed in the animals during the study.
The body weights of all rabbits were considered to be within the normal range of variability.
No intercurrent deaths occurred throughout the study.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the Draize classification criteria the test substance is considered to be a “mild- irritant” to rabbit skin (P.I.I. = 1.22) in the primary skin irritation potential test performed in compliance with GLP and according to the OECD 404 guideline.

Executive summary:

The primary skin irritation potential of test substance was investigated in compliance with GLP and according to the OECD 404 guideline. The animals were treated by topical semi-occlusive application of 0.5 g to the intact shaved flank of 3 young adult New Zealand White rabbits. The duration of treatment was 4 hours. The scoring of skin reactions was performed 1, 24, 48, 72 hours, 1, 2 and 3 weeks after removal of the dressing. The primary irritation index was calculated by totalling the mean cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points.

The primary irritation index was 1.22. At observations 1 hour after patch removal, very slight oedema (score 1) was observed in 2 animals. Oedema persisted in 1 animal and was observed at the 24- and 48 hour observations. Very slight erythema (score 1) was recorded in all animals from 1 hour after patch removal and at all subsequent observations up to and including the 1 week observations. At the 2 week observations very slight erythema (score 1) was still observed in 2 animals. Dry cracked skin surface was observed in all animals at the 1 week observation and in 1 animal at the 2 week observation. As signs of irritation were observed after 2 weeks, irreversibility could not be determined, and the study was therefore extended by 1 week. All animals fully recovered and showed no local signs at the 3 weeks observation.

The study was terminated after the 3 weeks observation as no signs were observed in the treated animals.

According to the Draize classification criteria the test substance is considered to be a “mild- irritant” to rabbit skin (P.I.I. = 1.22) in the primary skin irritation potential test performed in compliance with GLP and according to the OECD 404 guideline.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Oct 2010 to 18 Oct 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

24 April 2002

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

April 29, 2004

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, B.5 (L 142)

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age/weight at dosing: Approximately 13 weeks / 3116-3161 g
- Housing: Individually housed in metal wire rabbit cages
- Acclimatisation period: 12 days
- Diet: Lap gr. diet for rabbit; ad libitum
- Water: Tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3°C
- Humidity: 30 - 70%
- Air changes: 15 - 20/hour
- Photoperiod: 12 hours of light/12 hours of dark

IN-LIFE DATES: 11 Oct 2010 to 18 Oct 2010

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: A volume of 0.1 mL

Duration of treatment / exposure:

Single treatment

Observation period (in vivo):

1, 24, 48, 72 hours and 1 week following treatment

Number of animals or in vitro replicates:

3 animals

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The eyes of the test animals were not washed out after application of test item.

OBSERVATIONS:
Assessment of ocular damage/irritation was made approximately 1, 24, 48, 72 hours and 1 week following treatment.
Additionally, to assess the presence of corneal damage, fluorescein staining was employed at the following time points after the standard observations have been made:
- At least 24 hours prior to instillation
- At 24 hours post dose
- All subsequent observation times points until Draize scores are zero
The duration of the observation period was sufficient to identify reversibility of changes. Any clinical signs of toxicity or signs of ill-health during the study were recorded.

SCORING SYSTEM:
Assessment of ocular damage/irritation was made approximately according to Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal: #2 and #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Remarks:

(discharge)

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal: #2 and #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Conjunctival redness (score 1), discharge (score 2) and chemosis (score 1) were observed in all animals at the 1 hour observations. Chemosis (score 1) remained in two animals at the 48 hour observation. Conjunctival redness (scores 1/2) persisted in all animals at the 24- and 48- hour observations, and in two animals at the 72 hour observation. One week after treatment no further signs were observed and all local signs had fully reversed in all animals.
Two animals showed a positive fluorescein stain 24 hours after treatment, which was no longer observed at the 48 hour observation.

Other effects:

- No deaths occurred.
- No systemic signs of toxicity were noted during the study.
- The body weights of the rabbits were considered to be within the normal range of variability.
- Immediately following instillation, slight initial pain (2 on a 0-5 scale) was observed in all animals.
- No abnormal findings were observed in the cornea or iris of any animal at any of the measurement intervals.

Table 1. Eye irritation scores of test item according to the Draize scheme

Rabbit number and sex
	Male #1					Male #2					Male #3
Time after treatment (h)	1	24	48	72	1 wk	1	24	48	72	1 wk	1	24	48	72	1 wk
CORNEA E = Degree of Opacity F = Area of Cornea involved	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
IRIS D	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
CONJUNCTIVAEA=Redness B = Chemosis C =Discharge	1 1 2	2 0 0	1 0 0	1 0 0	0 0 0	1 1 2	1 1 0	1 0 0	1 0 0	0 0 0	1 1 2	2 1 0	1 0 0	0 0 0	0 0 0
Score (A+B+C) x 2	8	4	2	2	0	8	4	2	2	0	8	6	2	0	0
Total Score	8	4	2	2	0	8	4	2	2	0	8	6	2	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

In a primary eye irritation study performend in compliance with GLP and according to OECD 405 guideline the test substance is not an eye irritant.

Executive summary:

In a primary eye irritation study performed in compliance with GLP and according to OECD 405 guideline, 0.1 mL of the test substance was instilled into the conjunctival sac of the left eye of each of a group of three adult male New Zealand White rabbits. Immediately after instillation of the test material, an assessment of the initial pain reaction was made. Assessment of ocular damage/irritation was made approximately 1, 24, 48, 72 hours and 1 week following treatment, according to a numerical evaluation (Draize JH, 1977). Using the numerical data obtained a modified version of the system described by Kay JH and Calandra JC, 1962 was used to classify the ocular irritancy potential of the test material.

Slight initial pain was observed in all animals. Conjunctival redness, discharge and chemosis were observed in all animals at the 1 hour observations. Chemosis remained in two animals at the 48 hour observation. Conjunctival redness persisted in all animals at the 24- and 48- hour observations, and in two animals at the 72 hour observation. One week after treatment no further signs were observed and all local signs had fully reversed in all animals. Two animals showed a positive fluorescein stain 24 hours after treatment, which was no longer observed at the 48 hour observation. The study was terminated after the 1 week observation. No clinical signs of systemic toxicity were observed in the animals during this study. No intercurrent deaths occurred during this study. The body weights of all rabbits were considered to be within the normal range of variability.

The test substance is not an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key studies. The in vitro data requirements were waived based on the availability of adequate in vivo data. Dosing with the test material in rabbits resulted in mild irritation to the skin and to the eye, but no sufficient effect for classification under CLP.

Skin irritation

The primary skin irritation potential of test substance was investigated in compliance with GLP and according to the OECD TG 404. The animals were treated by topical semi-occlusive application of 0.5 g to the intact shaved flank of 3 young adult New Zealand White rabbits. The duration of treatment was 4 hours. The scoring of skin reactions was performed 1, 24, 48, 72 hours, 1, 2 and 3 weeks after removal of the dressing. The primary irritation index was calculated by totalling the mean cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points.

The primary irritation index was 1.22. At observations 1 hour after patch removal, very slight oedema (score 1) was observed in 2 animals. Oedema persisted in 1 animal and was observed at the 24- and 48 hour observations. Very slight erythema (score 1) was recorded in all animals from 1 hour after patch removal and at all subsequent observations up to and including the 1 week observations. At the 2 week observations very slight erythema (score 1) was still observed in 2 animals. Dry cracked skin surface was observed in all animals at the 1 week observation and in 1 animal at the 2 week observation. As signs of irritation were observed after 2 weeks, irreversibility could not be determined, and the study was therefore extended by 1 week. All animals fully recovered and showed no local signs at the 3 weeks observation. The study was terminated after the 3 weeks observation as no signs were observed in the treated animals.

According to the Draize classification criteria the test substance is considered to be a “mild- irritant” to rabbit skin (P.I.I. = 1.22) in the primary skin irritation potential test performed in compliance with GLP and according to the OECD TG 404.

One supporting study is available (Ullmann & Sachsse 1979) which was conducted according to AFDO 1959 guidelines and did not follow GLP. The test substance was administered to male and female rabbits as a single dermal dose of 0.5 mL. The erythema and edema scores over 24, 48 and 72 hours in animal #1 were 0. The erythema and edema scores over 24, 48 and 72 hours in animal #2 were 0.66 and 0.33 which recovered within 72 and 48 hours, respectively. The erythema and edema scores over 24, 48 and 72 hours in animal #3 were 1 which recovered within 4 and 7 days, respectively. The erythema and edema scores over 24, 48 and 72 hours in animal #4 were 0.66 and 0.33 which recovered within 48 hours, respectively. The erythema and edema scores over 24, 48 and 72 hours in animal #5 were 1.66 and 1.33 which recovered within 4 days, respectively. The erythema and edema scores over 24, 48 and 72 hours in animal #6 were 1.33 and 1 which recovered within 4 and 7 days, respectively. The test substance was found to cause a slight irritation and the calculated primary irritation score was 1.7. 

Eye Irritation

In the key study (Tavazsi 2010), performed in compliance with GLP and according to OECD TG 405, 0.1 mL of the test substance was instilled into the conjunctival sac of the left eye of each of a group of three adult male New Zealand White rabbits. Immediately after instillation of the test material, an assessment of the initial pain reaction was made. Assessment of ocular damage/irritation was made approximately 1, 24, 48, 72 hours and 1 week following treatment, according to a numerical evaluation (Draize JH, 1977). Using the numerical data obtained a modified version of the system described by Kay JH and Calandra JC, 1962 was used to classify the ocular irritancy potential of the test material.

Slight initial pain was observed in all animals. Conjunctival redness, discharge and chemosis were observed in all animals at the 1 hour observations. Chemosis remained in two animals at the 48 hour observation. Conjunctival redness persisted in all animals at the 24- and 48- hour observations, and in two animals at the 72 hour observation. One week after treatment no further signs were observed and all local signs had fully reversed in all animals. Two animals showed a positive fluorescein stain 24 hours after treatment, which was no longer observed at the 48 hour observation. The study was terminated after the 1 week observation. No clinical signs of systemic toxicity were observed in the animals during this study. No intercurrent deaths occurred during this study. The body weights of all rabbits were considered to be within the normal range of variability.

Three supporting studies are available which support the findings of the key study. The studies followed differnet guidelines and were not conducted according to GLP. In the first study (Ullmann & Sachsse 1978), the test substance was administered to male and female rabbits as a single dose to the eye of 0.5 mL. The cornea opacity score and iris score over 24, 48 and 72 hours in all animals with rinsed eyes was 0. The conjunctivae score over 24, 48 and 72 hours in all animals with rinsed eyes except for one animal was 0. That differing animal (#5) has a score of 6/20 at 24 hours which recovered within 48 hours. The test material was found to cause a minimal irritation when applied to the rabbit eye mucosa. Rinsing the eyes following instillation was of very good effect. In the second study (Kuhn 1998), albino rabbits were doses with 0.1 mL test material. The cornea opacity score and iris score over 24, 48 and 72 hours in all animals with washed eyes was 0. The conjunctivae score over 24, 48 and 72 hours in all three animals with washed eyes ranged from 0-4 (maximum score possible is 20) and all effects recovered within 4 days. The test substance was considered to be not irritating to the eyes of rabbits. In the third study (Gillis 1990), rabbits were doses with 100 mg test substance. Eye irritation evaluation in the nonwash animals beginning 24 hours after dosing found chemosis, discharge, and redness clear in all animals at 48 hours; corneal opacity was clear by 72 hours. For the wash animals, all effects were clear by 48 hours. Average irritation scores for the nonwash animals were 8.7, 3.3, and 0.0 at 24, 48, and 72 hours, respectively. Average irritation scores for the wash animals were 2.0, 0.0, and 0.0 at 24, 48, and 72 hours, respectively. The test substance was considered to be not irritating to the eyes of rabbits.

Justification for classification or non-classification

Based on the available information, classification of the test substance for skin and eye irritation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.