3,5-dimethylpyrazole

Endpoint summary

Description of key information

SKIN IRRITATION

Key study; Parcell (1994b), performed to GLP, OECD 404 and EU method B.4, Klimisch 1, not irritating.

Supporting study; Wallace (1976b), non-GLP study performed to sound scientific principles, Klimisch 2, not irritating.

EYE IRRITATION

WoE; Parcell (1994c), performed to GLP and EU method B.5, Klimisch 1, not irritating.

WoE; Hutchinson (2002b) performed to GLP, OECD 405, EU Method B.5 and EPA OPPTS 870.2440, Klimisch 1, not irritating.

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:

5th - 8th April 1994.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: shorter observation period.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

not applicable

Principles of method if other than guideline:

The observation period was only for 4 days.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Froxfield Ltd. (U.K), Petersfield, Hampshire, England.
- Age at study initiation: 11 to 13 weeks.
- Weight at study initiation: 2.6 to 3.1 kg.
- Housing: Individually in plastic cages with perforated floors.
- Diet (e.g. ad libitum): Standard laboratory diet SDS Stanrab (P) Rabbit Diet, as libitum.
- Water (e.g. ad libitum): Drinking water, ad libitum.
- Acclimation period: Period not reported, however animals were allowed to acclimatise to the test conditions prior to testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 19ºC.
- Humidity (%): 30-70%.
- Air changes (per hr): 19 charges per hour.
- Photoperiod (hrs dark / hrs light): 12 hour of artificial light (0700-1900) in each 24 hour period.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

Single dose of 0.5g

Duration of treatment / exposure:

Four hours.

Observation period:

Four days.

Number of animals:

Three

Details on study design:

TEST SITE
- Area: Dorso-lumbar region.
- Area of exposure: Approximately 25 mm x 25 mm.
- Site was prepared 24 hours prior to exposure.
- Type of wrap if used: gauze pad and an 'Elastoplast' dressing.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes with warm water (30 - 40 ºC)
- Time after start of exposure: 4 hours, the end of the exposure period.

SCORING SYSTEM: Draize scale (1977) as seen in table 1 in the field "any other information on material and methods incl. tables".

OBSERVATIONS:
- Clinical signs: animals were observed daily for signs of ill health or toxicity.
- The dermal response was scored at the following time point; 60 mins after removal of dressing and 24, 48 and 72 hours after exposure.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No reaction observed at any time point.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No reaction observed at any time point.

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No reaction observed at any time point.

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No reaction observed at any time point.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No reaction observed at any time point.

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: No reaction observed at any time point.

Other effects:

Clinical signs: no signs of toxicity or ill health in any animal during the observation period.
Dermal response: no dermal reaction was observed in any of the test animals.

Table 2. Dermal Response

		Observation Time
Rabbit Number	Observation	60 min	Day 2	Day 3	Day 4
366	Erythema	0	0	0	0
	Oedema	0	0	0	0
367	Erythema	0	0	0	0
	Oedema	0	0	0	0
368	Erythema	0	0	0	0
	Oedema	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

There was no dermal response or signs of toxicity were observed in any of the test animals after a 4 hour exposure.

Executive summary:

In a GLP compliant study performed according to the standardised guideline EU Method B.4 the dermal response caused by exposure to the test material was determined. Three New Zealand White rabbits were exposed to the test material for 4 hours; the reaction was scored according to the Draize scale (1997), over a 4 day observation period. No dermal response or clinical signs of toxicity were observed in any of the test animals. It is this lack of any reaction which is thought to be the reason the observations were terminated after only 4 days.

The skin irritation study indicates that the test material does not require classification according to Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 April 2002 to 16 April 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adults
- Weight at study initiation: On dose day individual animals weighted 3.99, 3.60 and 3.81 kg.
- Housing: Individually in stainless steel cages, approximately 77 x 70 x 48 cm.
- Diet: The feed was supplied ad libitum throughout the study, supplemented with hay twice weekly.
- Water: Water was supplied ad libitum throughout the study from the domestic main supply.
- Analysis for significant contaminants was performed on both the diet and water supplied to the test animals. Both were considered not to contain any additional substances in sufficient concentrations to have any influence on the outcome of the study.
- Acclimation period: At least 5 days prior to study initiation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Mean minimum and maximum temperatures were 21ºC and 20ºC, respectively.
- Humidity (%): Ranged from 28 to 37% with a mean humidity of 32%.
- Air changes (per hr): Minimum of 15 air charges per hour.
- Photoperiod (hrs dark / hrs light): 12 hr light/dark cycle (light 0700-1900 hrs).

Vehicle:

unchanged (no vehicle)

Controls:

other: left eyer served as control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 65.2, 64.7 and 63.5 mg was instilled into the eye of rabbits numbered 1, 2 and 3. These are the weight equivalent to 0.1 mL.

Duration of treatment / exposure:

Single dose

Observation period (in vivo):

The initial observation period was 72 hours, which was extended to 8 Days (7 days after exposure) for two rabbits in order to assess reversibility.

Number of animals or in vitro replicates:

3

Details on study design:

APPLCATION OF THE TEST MATERIAL
Ocular examination: Animals were examined before application of the test material and no ocular defects were detected.
Application: The test material was placed into the right eye by gently pulling the lower eyelid away from the eyeball to form a sac into which the test material was dropped. The lid was held together gently for 1 to 2 seconds. One rabbit was treated first to assess the severity of the reaction over 24 hours. As none were noted, the remaining two rabbits were treated in the same manner.
Control: The left eye remained untreated to serve as a control, which as examined in the same manner as the treated eye.

SCORING SYSTEM: Draize scale, see Table 1.
Observation intervals: 1, 24, 48 and 72 hrs and then Day 8.
Viability: Checked twice daily.
Clinical observations: All animals were examined for any reaction to treatment. The onset, intensity and duration of any reactions were recorded.
Body weight: Recorded on Day 0 (day of exposure).

TOOL USED TO ASSESS SCORE: A hand held magnifier and pen torch was used where necessary. To assess corneal damage, fluorescein sodium ophthalmic solution (BP)(2%) was used.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.75

Max. score:

3

Reversibility:

fully reversible within: 8 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.25

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.75

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.5

Max. score:

2

Reversibility:

fully reversible within: 8 days

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.5

Max. score:

3

Reversibility:

fully reversible within: 8 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.25

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.75

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.75

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

- Between 1 and 48 hours after treatment corneal opacity was noted in one animal (easily discernible translucent area covering between three quarters and the whole of the cornea). By 72 hours the effect was notably reduced to scattered or diffuse areas of opacity covering between a quarter and a half of the cornea.
- Conjunctival redness and chemosis was noted in all three animals up to 72 hours after instillation.
- Moderate discharge was noted in one animal only 1 hour after instillation and slight to moderate discharge was noted in a second animal up to 48 hours after application of the test material.
- No responses were noted in the control eye.

Table 2. Reaction Scores for Treated and Control Eyes

Area of Eye			Animal No. 1					Animal No. 2					Animal No. 3
			Time after instillation of the Test Material (hours/days)
			1hr	24hr	48hr	72hr	Day 8	1hr	24hr	48hr	72hr	Day 8	1hr	24hr	48hr	72hr
Test Eye
Cornea:	Degree/Opacity		0	0	0	0	0	2	2	2	1	0	0	0	0	0
	Area/Opacity		0F	0	0	0	0	4F	4F	4F	2F	0F	0F	0	0	0
Iris			0	0	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctivae:	Redness		2	2	2	1	0	2	2	1	1	0	1	1	1	0
	Chemosis		2	1	1	1	0	2	1	1	1	0	2	1	0	0
Discharge			2	0	0	0	0	1	2	1	0	0	0	0	0	0
Control Eye
Cornea:		Degree/Opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Area/Opacity	0F	0	0	0	0	0F	0F	0F	0F	0F	0F	0	0	0
Iris			0	0	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctivae:		Redness	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Chemosis	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Discharge			0		0	0	0	0	0	0	0	0	0	0	0	0

F = Fluorescein stanied

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, the test material is considered to be non-irritating to the eye in rabbits. Exposure to the test material caused some mild reactions, however these reactions were not severe enough to permit classification. Corneal opacity was noted in one animal between up to 72 hrs post application, conjunctivae redness and discharge was observed in all three animals, however these reactions were mild and fully reversible by Day 8.

Executive summary:

The irritation potential of the test material was assessed in an acute eye irritation study, performed to GLP and in line with the standardised guidelines OCED 405, EU Method b.5 and EPA OPPTS 870.2400.

The weight equivalent to 1 mL of the test material was applied to the right eye of three New Zealand White rabbits, the contra-lateral eye serving as an untreated control. The initial observation period was 72 hours, which was extended to 8 Days (7 days after exposure) for two rabbits in order to assess the reversibility of any reactions.

Exposure to the test material caused some mild reactions, however these reactions were not severe enough to permit classification. Corneal opacity was noted in one animal between up to 72 hrs post application, conjunctivae redness and discharge was observed in all three animals, however these reactions were mild and fully reversible by Day 8.

Under the conditions of the test, the test material is considered to be non-irritating to the eye in rabbits.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11th - 25th April 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: observation period

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Principles of method if other than guideline:

The observation period was only for 7 days.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Froxfield Ltd (UK)., Petersfield, Hampshire, England.
- Age at study initiation: 13 to 15 weeks.
- Weight at study initiation: 3.0 to 3.5 kg.
- Housing: Individually in plastic cages with perforated floors.
- Diet (e.g. ad libitum): Standard laboratory diet SDS Stanrab (P) Rabbit Diet, as libitum. Diet was not analysed.
- Water (e.g. ad libitum): Drinking water, ad libitum. Routine weekly water analysis.
- Acclimation period: Period not reported, however animals were allowed to acclimatise prior to testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ºC.
- Humidity (%): 30 - 70%
- Air changes (per hr): ~ 19 charges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of artificial light (0700 - 1900) in each 24 hour period.

Vehicle:

unchanged (no vehicle)

Controls:

other: contralateral eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): 60 mg of test material.

Duration of treatment / exposure:

single exposure

Observation period (in vivo):

Seven days post exposure.
Intervals of observations: 1, 24, 48 and 72 hours, day 4 and day 7.

Number of animals or in vitro replicates:

three

Details on study design:

PRE-TREATMENT EYE EXAMINATION:
- Animals eyes were first examined for pre-existing corneal damage, iridial or conjunctival inflammation, only those free of damage were used for the study.

METHOD:
- One animal was treated ahead of the others, to assess the severity of the reaction.
- The test material was placed into the inverted eyelid of each animal.
- The eyelid was held together for one second, before the animal was released.

REMOVAL OF TEST SUBSTANCE:
- Washing: Not performed

SCORING SYSTEM: Draize scale (1977), which can be seen in table 1.

OTHER OBSERVATIONS:
- Clinical signs, all animals were examined daily for ill health or signs of toxicity.

TOOL USED TO ASSESS SCORE: handheld light.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of all three animals

Time point:

24/48/72 h

Score:

0.77

Max. score:

4

Reversibility:

fully reversible

Remarks:

by day 4

Irritation parameter:

iris score

Basis:

mean

Remarks:

of all three animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

of all three animals

Time point:

24/48/72 h

Score:

0.77

Max. score:

3

Reversibility:

fully reversible

Remarks:

by 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of all three animals

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Remarks:

by 48 hours

Other effects:

Clinical signs - there were no signs of toxicity or ill health in any rabbit during the observation period.

Table 2. Ocular Reactions Observed after Installation of the test Material

Rabbit number	Region of eye	Observation Time Interval
		1 hour	24 hours	48 hours	72 hours	Day 4	Day 7
350 *	Cornea	3	1	1	1	0	0
	Iris	0	0	0	0	0	0
	Conjunctive Redness	2	2	1	0	0	0
	Conjunctiva Chemosis	3	1	0	0	0	0
381	Cornea	aD	1	1	0	0	0
	Iris	0	0	0	0	0	0
	Conjunctive Redness	2	1	1	0	0	0
	Conjunctiva Chemosis	3	1	0	0	0	0
382	Cornea	D	1	1	0	0	0
	Iris	0	0	0	0	0	0
	Conjunctive Redness	2	1	1	0	0	0
	Conjunctiva Chemosis	2	1	0	0	0	0

* = Pilot animal

a = Loss of corneal epithelia cells

D = Dulling

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the ocular response shown by the three rabbits, the test material is not irritating to the eye. A mild initial reaction was observed, however this was fully reversible within 4 days.

Executive summary:

In a GLP compliant study performed according to the standardised guideline EU Method B.5, the eye irritation caused by exposure to the test material was determined in the New Zealand White rabbit. Three rabbits were exposed to 60 mg of the test material in one eye, using the contralateral as a control. The ocular response was observed and scored according to the Draize scale (1977) over 7 days. Under the conditions of the test the test material did not cause a strong enough reaction to warrant classification according to Regulation (EC) 1272/2008. A mild initial reaction was observed, however this was fully reversible within 4 days and no clinical signs of toxicity were seen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:

The key study for skin irritation (Parcell, 1994b) was determined in a GLP compliant study performed in accordance with the standardised guidelines OECD 404 and EU Method B.4. The study was performed to a good standard with a sufficient level of detail to assess the quality of the data presented. The study was assigned a reliability score of 1, in accordance with Klimisch (1997) and considered suitable to fulfil the data requirement. The test material was determined to be non-irritating.

A supporting study (Wallace, 1976b) also determined the test material to be non-irritating in a non-GLP study performed to sound scientific principles. The study was performed to a good standard with a sufficient level of detail to assess the quality of the data presented. The study was assigned a reliability score of 2, in accordance with Klimisch (1997).

Justification for selection of eye irritation endpoint:

This endpoint has been addressed on a weight of evidence basis and thus one study cannot be selected over the other. Parcell, (1994c) and Hutchinson (2002b) were performed according to GLP and in line with standardised guidelines. Both studies were performed to a good standard with a sufficient level of detail to assess the quality of the data presented. They were assigned a reliability score of 1, in accordance with Klimisch (1997) and considered suitable to fulfil the data requirement. The test material was determined to be non-irritating.

Justification for classification or non-classification

With respect to the information available, the test material does not meet the criteria for classification as either an eye or skin irritant in accordance with Regulation (EC) No. 1272/2008.