Registration Dossier

Administrative data

Description of key information

- Key study: skin irritation (in vivo; OECD 405; New Zealand White rabbits; semiocclusive): slightly irritating;

- Supporting: skin irritation (in vitro; OECD 439): irritating;
- Supporting: skin corrosion (in vitro; Corrositex Assay; no guideline available): non-corrosive.
- Key study: eye irritation (in vivo; OECD 405; New Zealand White rabbits, undiluted): 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:
2013-11-26 to 2013-12-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance, Denver, PA
- Age at study initiation: 25 to 26 weeks
- Weight at study initiation: between 2.9 and 3.8 kg
- Housing: individually in suspended stainless steel cages
- Diet (e.g. ad libitum): PMI Nutrition International Certified Rabbit Chow No. 5322 was provided ad libitum throughout the study except during designated procedures. To avoid potential gastrointestinal disturbances, food was withheld for approximately 24 to 48 hours after receipt. Food was then gradually increased over a 3-day period.
- Water (e.g. ad libitum): Municipal tap water following treatment by reverse osmosis and ultraviolet irradiation was available ad libitum throughout the study.
- Acclimation period: at least 7 days before the first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 44-49
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Nov 2013 (day of arrival) To:
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
The test substance was administered as received (pure). Dose calculations were not corrected for purity.
Duration of treatment / exposure:
- animal 1: 3 min, 1h and 4hours
- animal 2: 4 hours
- animal 3: 4 hours
Observation period:
For the 3-minute exposure: Immediately after patch removal, 1 hour after patch removal, and 24, 48, and 72 hours after patch application.
For the 1-hour exposure: Immediately after patch removal, 1 hour after patch removal, and 24, 48, and 72 hours after patch application.
For the 4-hour exposure: 1 hour after patch removal, and 24, 48, and 72 hours after patch application.
If dermal irritation persisted at any test site, the observation period was extended for the affected animals up to 21 days.
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: approximately 1 inch x 1 inch
- Type of wrap if used: The gauze patches were held in contact with the skin at the cut edges with a nonirritating tape. Removal and ingestion of the test substance were prevented by placing an elastic wrap over the trunk and test area (semi-occlusive binding).

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test substance was removed using gauze moistened with deionized water followed by dry gauze.
- Time after start of exposure: immediately after patch removal.

SCORING SYSTEM:
EPA-FIFRA Dermal Irritation Descriptive Classification:
The 1- (or initial observation), 24-, 48- and 72-hour erythema and oedema scores for all animals were added and the total divided by the number of test sites (5 (= animal 1(3 test sites: 3-min, 1-hour, 4-hour) + animal 2 (1) + animal 3(1))) x 4 (= number of observation time points: 1-hour, 24-hour, 48-hour and 72-hour) to yield the Primary Irritation Index (P.I.I.). The calculated Primary Irritation Index (P.I.I.) was classified according to the Dermal Irritation Descriptive Classification (1).

EEC Dermal Evaluation Criteria:
The 24-, 48- and 72-hour scores were added separately for erythema and oedema. For a group of three animals, the calculated mean scores were expressed individually. The resulting appropriate Primary Irritation Indices (P.I.I.) was classified according to the EEC Dermal Evaluation Criteria (2).

References:
1. Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals-Addendum 3 on Data Reporting, U.S. Environmental Protection Agency, 1988.
2. Commission of European Communities, Official Journal of European Communities, Annex VI, General Classification and Labeling Requirements for Dangerous Substances and Preparations, No. L 225/263-314, August 2001.
 
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
animal #1
Time point:
other: 3-min exposure
Score:
0
Max. score:
0
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: very slight erythema was noted on Day 14, which subsequently resolved completely by Day 21
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
animal #1
Time point:
other: 1-hour exposure
Score:
0.75
Max. score:
1
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: The dermal irritation was not observed on Day 7, but was observed again on Day 14.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Remarks:
of 3 animals
Time point:
other: 4-hour exposure
Score:
1.33
Max. score:
2
Reversibility:
not fully reversible within: 14 days
Remarks on result:
other: The dermal irritation was not observed in 1/3 test sites on Day 7, but was again observed in that animal on Day 10. Dermal irritation did not resolve completely in the remaining 2 test sites.
Irritation parameter:
erythema score
Remarks:
mean from gradings at 24, 48 and 72-hour observation time points
Basis:
animal #1
Time point:
other: 4-hour exposure
Score:
2
Max. score:
2
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: the score of 1 for erythema was still present at day 21 post-exposure
Irritation parameter:
erythema score
Remarks:
mean from gradings at 24, 48 and 72-hour observation time points
Basis:
animal #2
Time point:
other: 4-hour exposure
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 21 days
Irritation parameter:
erythema score
Remarks:
mean from gradings at 24, 48 and 72-hour observation time points
Basis:
animal #3
Time point:
other: 4-hour exposure
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 21 days
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 1-hour observation time point
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
edema score
Remarks:
mean from gradings at 24, 48 and 72-hour observation time points
Basis:
animal #1
Time point:
other: 4-hour exposure
Score:
0
Max. score:
0
Remarks on result:
other: no irritation responses
Irritation parameter:
edema score
Remarks:
mean from gradings at 24, 48 and 72-hour observation time points
Basis:
animal #2
Time point:
other: 4-hour exposure
Score:
0
Max. score:
0
Remarks on result:
other: no irritation responses
Irritation parameter:
edema score
Remarks:
mean from gradings at 24, 48 and 72-hour observation time points
Basis:
animal #3
Time point:
other: 4-hour exposure
Score:
0
Max. score:
0
Remarks on result:
other: no irritation responses
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
3-Minute Exposure
Exposure to the test substance produced no erythema or oedema through 72 hours post-dose; however, very slight erythema was noted at the single test site on Day 14, which subsequently resolved completely by Day 21 (Table 1).
 
1-Hour Exposure
Exposure to the test substance produced very slight erythema at the single test site immediately after patch removal (Table 2). The dermal irritation was not present at the 1-hour scoring interval, but was present again at the 24-, 48-, and 72-hour scoring intervals. The dermal irritation was not observed on Day 7, but was observed again on Day 14. Day 21 scoring revealed no irritation. Additional dermal findings included desquamation.
 
4-Hour Exposure
Exposure to the test substance produced very slight erythema at 3/3 test sites and very slight oedema at 1/3 test sites by the 1-hour scoring interval (Table 3). Well-defined erythema was observed in 1/3 test sites at the 24-, 48-, and 72-hour scoring intervals. The dermal irritation was not observed in 1/3 test sites on Day 7, but was again observed in that animal on Day 10. Dermal irritation did not resolve completely in the remaining 2 test sites. Additional dermal findings included blanching (focal and/or pinpoint areas up to 10% of the test site) in 1/3 test sites and desquamation in 3/3 test sites.
Other effects:
No other effects were reported in treated animals.

Table 1. Individual dermal irritation scores (3 -min exposure; animal 1)

Animal No. /Sex/ Body weight (kg)

Scoring Interval

Erythema

Oedema

5810/M

2.903

Patch removal

0

0

1 hour

0

0

24 hours

0

0

48 hours

0

0

72 hours

0

0

7 days

-

-

10 days

-

-

14 days

1

0

21 days

0

0

Primary Irritation Index = 0.00 (Non-irritant)

Table 2. Individual dermal irritation scores (1-hour exposure; animal 1)

Animal No. /Sex

Scoring Interval

Erythema

Oedema

Comments

5810/M

Patch removal

1

0

 

1 hour

0

0

 

24 hours

1

0

 

48 hours

1

0

 

72 hours

1

0

 

7 days

0

0

DES

10 days

0

0

DES

14 days

1

0

DES

21 days

0

0

 

DES - desquamation

Primary Irritation Index = 0.75 (Slight irritant)

PII = ((Summe of erythema and oedema scores at 1, 24, 48 and 72 hours) / ( 1 (Number of test sites) x 4 (Number of scoring intervals))

Table 3. Individual dermal irritation scores (4-hour exposure; animal 1, 2 and 3)

Animal No. /Sex/ Body weight (kg)

Scoring Interval

Erythema

Oedema

Comments

5810/M

Patch removal

1

0

 

1 hour

1

1

 

24 hours

2

0

BLA-1

48 hours

2

0

BLA-1

72 hours

2

0

BLA-1

7 days

1

0

DES

10 days

1

0

DES

14 days

1

0

DES

21 days

1

0

DES

 

 

 

 

5811/M
3.784

Patch removal

1

0

 

1 hour

1

0

 

24 hours

1

0

 

48 hours

1

0

 

72 hours

1

0

 

7 days

1

0

 

10 days

1

0

DES

14 days

1

0

DES

 

 

 

 

5812/M
3.133

Patch removal

1

0

 

1 hour

1

0

 

24 hours

1

0

 

48 hours

1

0

DES

72 hours

1

0

 

7 days

0

0

 

10 days

1

0

DES

14 days

1

0

DES

DES: desquamation

BLA-1: blanching

Primary Irritation Index = 1.33 (Slight irritant)

PII = ((Sum of erythema and oedema scores at 1, 24, 48 and 72 hours) / ( 3 (Number of test sites) x 4 (Number of scoring intervals))

Interpretation of results:
slightly irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: according to Primary Irritation Index
Conclusions:
Under the conditions of the test (4 hour exposure), the test substance is considered to be a slight irritant to the skin of the rabbit. The calculated Primary Irritation Index for the test substance was 1.33.
According to the EEC Dermal Evaluation Criteria, the test substance is considered to be a nonirritant for erythema and oedema
Executive summary:

The objective of this study was to assess the corrosive effects of the test substance when given as a single dermal administration to rabbits. Each of 3 rabbits received a 0.5 mL dose of the test substance as a single dermal application. The dose was held in contact with the skin under a semi-occlusive binder for exposure periods of 3 minutes, 1 hour, and 4 hours for 1 animal and exposure periods of 4 hours for 2 animals. Following the completion of each exposure period, the binder was removed and the remaining test substance was wiped from the skin using gauze moistened with deionized water followed by dry gauze. Test sites were subsequently examined and scored for dermal irritation for up to 21 days following patch removal.

 

3-Minute Exposure

Exposure to the test substance produced no erythema or oedema through 72 hours post-dose; however, very slight erythema was noted at the single test site on Day 14, which subsequently resolved completely by Day 21.

 

1-Hour Exposure

Exposure to the test substance produced very slight erythema at the single test site immediately after patch removal. The dermal irritation was not observed at the 1-hour scoring interval, but was present again at the 24-, 48-, and 72-hour scoring intervals. The dermal irritation was not observed on Day 7, but was observed again on Day 14. Day 21 scoring revealed no irritation. Additional dermal findings included desquamation.

 

4-Hour Exposure

Exposure to the test substance produced very slight erythema at 3/3 test sites and very slight oedema at 1/3 test sites by the 1-hour scoring interval. Well-defined erythema was observed in 1/3 test sites at the 24-, 48-, and 72-hour scoring intervals. The dermal irritation was not observed in 1/3 test sites on Day 7, but was again observed in that animal on Day 10. Dermal irritation did not resolve completely in the remaining 2 test sites. Additional dermal findings included blanching (focal and/or pinpoint areas up to 10% of the test site) in 1/3 test sites and desquamation in 3/3 test sites.´

 

Conclusion

Under the conditions of the test (4 hour exposure), the test substance is considered to be a slight irritant to the skin of the rabbit. The calculated Primary Irritation Index (P.I.I) for the test substance was 1.33.

According to the EEC Dermal Evaluation Criteria, the test item is considered to be a nonirritant for erythema and oedema as listed below:.

Animal No./Sex

Erythema

Edema

P.I.I.

3-Minute Exposure

5810/M

0.00

0.00

0.00 - Nonirritant

Irritation Rating

Nonirritant

Nonirritant

1-Hour Exposure

5810/M

1.00

0.00

0.75 - Slight irritant

Irritation Rating

Nonirritant

Nonirritant

4-Hour Exposure

5810/M

2.00

0.00

1.33 - Slight irritant

5811/M

1.00

0.00

5812/M

1.00

0.00

Irritation Rating

Nonirritant

Nonirritant

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
2014-02-10 to 2014-03-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute 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
- Source: Covance, Denver, PA.
- Age at study initiation: 29 weeks
- Weight at study initiation: between 2.9 kg to 3.2 kg at initiation of dosing
- Housing: individually in suspended stainless steel cages
- Diet (e.g. ad libitum): ad libitum (PMI Nutrition International Certified Rabbit Chow No. 5322)
- Water (e.g. ad libitum): ad libitum (Municipal tap water following treatment by reverse osmosis and ultraviolet irradiation)
- Acclimation period: at least 12 days before the first day of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 69°F to 72°F (21°C to 22°C)
- Humidity (%): 37 - 43
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 2013-02-11 To: 2013-03-10
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
single exposure
Observation period (in vivo):
1, 24, 48, and 72 hours and up to 10 days after dosing
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no
SCORING SYSTEM: according to the Ocular Grading System (based on Draize) (see attached pdf)

TOOL USED TO ASSESS SCORE: fluorescein
The eyes were macroscopically examined with the aid of an auxiliary light source for signs of irritation at 1, 24, 48, and 72 hours and up to 10 days after dosing according to the Ocular Grading System presented above which is based on Draize. Following macroscopic observations at the 24-hour scoring interval, the fluorescein examination procedure was repeated on all test and control eyes and any residual test substance was gently rinsed from the eye at that time using physiological saline. If fluorescein findings were noted at 24 hours, a fluorescein exam was conducted on the affected eyes at each subsequent interval until a negative response was obtained and/or until all corneal opacity had cleared.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 10 days
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 10 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 10 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2.67
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 10 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
Exposure to the test article produced corneal opacity in 1/3 test eyes by the 24-hour scoring interval and complete resolution occurred by the Day 7 scoring interval. Iritis was observed in 1/3 test eyes by the 1-hour scoring interval and in 2/3 test eyes by the 24-hour scoring interval. Complete resolution of the iritis occurred in 1/3 test eyes by the Day 7 scoring interval and in 2/3 test eyes by the Day 10 scoring interval. Conjunctivitis (redness, swelling, and/or discharge) occurred in 3/3 test eyes by the 1-hour scoring interval and complete resolution occurred in 1/3 test eyes by the Day 7 scoring interval and in 2/3 test eyes by the Day 10 scoring interval. An additional ocular finding of neovascularization (2/3 test eyes) was noted during the study.
Other effects:
Decreased faecal output, including reduced faecal size and absence of faeces, was observed in all animals on at least 1 day during the study. Animals were treated with buprenorphine, an opioid, from Days 1 through 5, and opioids are known to cause decreased faecal output. Therefore, the decreased faecal output is likely an effect of the buprenorphine treatment.

Table 1. Individual ocular irritation scores

Animal No. /sex/ body weight (kg)

 

Cornea

Iris

Conjunctivae

Total

Test Eye

Control Eye

Interval

O

A

OxAx5

I

Ix5

R

S

D

(R+S+D) x 2

 

Fluorescein examination

Secondary ocular findings

Fluorescein examination

Secondary ocular findings

5981/M/

3.241

1 hour

0

0

0

0

0

2

3

2

14

14

 

 

 

 

24 hours

2

1

10

1

5

3

2

2

14

29

FAO

 

NFE

 

48 hours

3

1

15

1

5

3

2

2

14

34

FAO

 

 

 

72 hours

1

1

5

1

5

3

2

2

14

24

FAO

 

 

 

day 7

0

0

0

1

5

2

0

0

4

9

 

 

 

 

day 10

0

0

0

0

0

0

0

0

0

0

 

 

 

 

5984/M/

2.861

1 hour

0

0

0

0

0

2

3

2

14

14

 

 

 

 

24 hours

0

0

0

1

5

2

2

1

10

15

NFE

 

NFE

 

48 hours

0

0

0

1

5

3

2

1

12

17

 

VAS-1

 

 

72 hours

0

0

0

1

5

3

2

1

12

17

 

VAS-3

 

 

day 7

0

0

0

0

0

0

0

0

0

0

 

VAS-1

 

 

5985/M/

3.057

1 hour

0

0

0

1

5

2

3

2

14

19

 

 

 

 

24 hours

0

0

0

1

5

2

1

1

8

13

NFE

 

NFE

 

48 hours

0

0

0

1

5

2

0

1

6

11

 

 

 

 

72 hours

0

0

0

1

5

2

0

1

6

11

 

VAS-1

 

 

day 7

0

0

0

1

5

1

0

0

2

7

 

VAS-4

 

 

day 10

0

0

0

0

0

0

0

0

0

0

 

VAS-1

 

 

O - opacity; A - Area of cornea involved; I - Iritis; R - redness; S - swelling; D - discharge;

FAO - Fluorescein dye retention associated with the area of corneal opacity;

NFE - No fluorescein retention was observed;

VAS - 1: Total area of vascularized corneal tissue was < 10 % of corneal surface (neovascularization - very slight);

VAS - 2: Total area of vascularized corneal tissue was > 10 % but < 25 % of corneal surface (neovascularization - mild);

VAS - 3: Total area of vascularized corneal tissue was > 25 % but < 50 % of corneal surface (neovascularization - moderate);

VAS - 4: Total area of vascularized corneal tissue was > 50 % of corneal surface (neovascularization - severe).

Table 2. Mean ocular score

1 hour

15.67

24 hours

19.00

48 hours

20.67

72 hours

17.33

day 7

5.33

day 10

0.00

 

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
According to the Kay and Calandra Evaluation Criteria, the test item is considered to be a moderate irritant to the ocular tissue of the rabbit.
The total ocular irritation score for the 24-, 48-, and 72-hour intervals was individually added for corneal opacity, iris lesion, conjunctival redness, and conjunctival oedema. According to the EEC Ocular Evaluation Criteria, the test substance is classified as non-irritant for corneal opacity, irritant for iris lesions, conjunctival redness and oedema. According to the CLP criteria, the test item is classified as a Category 2 irritant to the eye (potential to induce reversible eye irritation).
Executive summary:

The objective of this study was to assess the irritant and/or corrosive effects of the test substance when given as a single ocular administration to rabbits. Each of 3 rabbits received a 0.1 mL dose of the test substance in the conjunctival sac of the right eye. The contralateral eye of each animal remained untreated and served as a control. Test and control eyes were examined for signs of irritation for up to 10 days following dosing.

Exposure to the test article produced corneal opacity in 1/3 test eyes by the 24-hour scoring interval and complete resolution occurred by the Day 7 scoring interval. Iritis was observed in 1/3 test eyes by the 1-hour scoring interval and in 2/3 test eyes by the 24-hour scoring interval. Complete resolution of the iritis occurred in 1/3 test eyes by the Day 7 scoring interval and in 2/3 test eyes by the Day 10 scoring interval. Conjunctivitis (redness, swelling, and/or discharge) occurred in 3/3 test eyes by the 1-hour scoring interval and complete resolution occurred in 1/3 test eyes by the Day 7 scoring interval and in 2/3 test eyes by the Day 10 scoring interval. An additional ocular finding of neovascularization (2/3 test eyes) was noted during the study. Decreased faecal output was observed in all animals. This observation, which is a known pharmacological effect of opioids, was likely due to the buprenorphine treatment that the animals received from Days 0 to 5. According to the Kay and Calandra Evaluation Criteria, the test substance is considered to be a moderate irritant to the ocular tissue of the rabbit. The total ocular irritation score for the 24-, 48-, and 72-hour intervals was individually added for corneal opacity, iris lesion, conjunctival redness, and conjunctival oedema. According to the EEC Ocular Evaluation Criteria, the test substance is classified as indicated below:

Animal No./Sex

Corneal Opacity

Iris Lesion

Conjunctival Redness

Conjunctival Edema

5981/M

2.00

1.00

3.00

2.00

5984/M

0.00

1.00

2.67

2.00

5985/M

0.00

1.00

2.00

0.33

Irritation Rating

Nonirritant

Irritant

Irritant

Irritant

According to the CLP criteria, the test substance is classified as a Category 2 irritant to the eye (potential to induce reversible eye irritation).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosivity (in vitro)

Prior to the in vivo and in vitro irritation testing, N-Butylpyrrolidone was evaluated with the Corrositex test method to determine its corrosive potential and to designate its Packing Group classification (in Vitro International, 2013). The Corrositex test is a standardized and reproducible method that can be employed to determine the potential corrosivity and determine the Packing Group classification of specified categories of chemical compounds under the hazardous materials transportation regulations administered by the U.S. Department of Transportation (DOT) and international dangerous goods codes. The Corrositex test predicts the in vivo corrosive potential of a chemical compound or mixture by using as an endpoint the time it takes for the chemical to permeate through or destroy a synthetic biobarrier. When the chemical has passed through this biobarrier, a visual change is produced in a proprietary Chemical Detection System (CDS).

To achieve this objective, the sample was subjected to a three-step testing process. First, a qualification test is done to insure that the test sample and the CDS reagent are compatible. This is achieved by placing either 150 μL of a liquid or 100 mg of a solid into an aliquot of the CDS reagent and observing it for the presence of any detectable change. If a physical or color change is observed, the sample is judged to be compatible with the detection solution and the remainder of the test is performed. The second step of the Corrositex test utilizes appropriate indicator solutions to permit categorization of the test sample as either a Category 1 or Category 2 material. Category 1 materials are typically strong acids/bases, while Category 2 materials are typically weak acids/bases. The third step in the test is performed by applying the test sample to the biobarrier. When the chemical permeates through or destroys the full thickness of this biobarrier, it comes into contact with the CDS which then undergoes a simple color change. This color change is visually observed and the time required for the color change to occur is recorded. The time required to destroy the biobarrier is recorded for four sample replicates and the mean of these replicates is utilized to designate the UN Packing Group classification as I (severe corrosivity; corrositex time is 0 - 3 min (Cat 1 and 2)), II (moderate corrosivity; corrositex time > 3 - 60 min (Cat 1) and > 3 - 30 min (Cat 2)), III (mild corrosivity; corrositex time is > 60 - 240 min (Cat 1) and > 30 - 60 min (Cat 2)), or Noncorrosive (NC; corrositex time is > 240 min (Cat 1) and > 60 min (Cat 2)). Positive and negative controls are analyzed concurrently to confirm the test’s validity. The results of this study indicated that the sample was compatible with the Corrositex system and was classified as a Category 2 material. The results obtained from the evaluation of four replicate samples were highly reproducible, demonstrating that a mean time of >60 minutes required to destroy the synthetic biobarriers. These findings lead to the designation of this sample as a non-corrosive.

Skin irritation (in vitro)

Since N-Butylpyrrolidone was not corrosive in the Corrositex test method, skin irritation potential of the test substance was determined in the Human Skin Model Test following OECD 439 Guideline (LAUS, 2012; Report No. 12092002G840). One valid experiment was performed. Three tissues of the human skin model EpiDerm™ were treated with N-Butylpyrrolidone for 60 minutes. 30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size (0.63 cm²; as indicated by supplier). DPBS-buffer was used as negative control, 5 % SDS-solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD (optic density) < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18 %). After the treatment with the test item, the relative absorbance values were reduced to 3.1 %. This value is well below the threshold for irritation potential (50 %). Therefore, N-Butylpyrrolidone is considered as irritant in the Human Skin Model Test. The study result supports findings in the in vivo test.

Skin irritation (in vivo)

According to the testing strategy outlined in OECD 404 Guideline (step 6), no further testing is required if a substance is irritating in a valid in vitro irritation test. Then, an appropriate classification as skin irritant should be required. However, the classification and labelling of the structurally related substances N-Methylpyrrolidone (CAS 872-50-4) and N-Ethylpyrrolidone (CAS 2687-91-4) differed for this endpoint: Cat 2, H315: Causes skin irritation (NMP) and non classified (NEP). Therefore, no definitive conclusion could be made whether or not NBP is irritating in vivo. Therefore an in vivo study was conducted.

N-Butylpyrrolidone was given as a single dermal administration to New Zealand White rabbits (OECD 404; Charles River Laboratories, 2014a; Report No. 20050801). Each of 3 rabbits received a 0.5 mL dose of the test substance as a single dermal application. The dose was held in contact with the skin under a semi-occlusive binder for exposure periods of 3 minutes, 1 hour, and 4 hours for 1 animal and exposure periods of 4 hours for 2 animals. Following the completion of each exposure period, the binder was removed and the remaining test substance was wiped from the skin using gauze moistened with deionized water followed by dry gauze. Test sites were subsequently examined and scored for dermal irritation for up to 21 days following patch removal.

 

At 3-minute exposure, the test substance did not produce erythema or oedema through 72 hours post-dose period. However, very slight erythema was noted at the single test site on Day 14, which subsequently resolved completely by Day 21.

 

After 1-hour exposure, the test substance produced very slight erythema at the single test site immediately after patch removal. The dermal irritation was not observed at the 1-hour scoring interval, but was present again at the 24-, 48-, and 72-hour scoring intervals. The dermal irritation was not observed on Day 7, but was observed again on Day 14. Day 21 scoring revealed no irritation. Additional dermal findings included desquamation.

 

After 4-hour exposure, the test substance produced very slight erythema at 3/3 test sites and very slight oedema at 1/3 test sites by the 1-hour scoring interval. Well-defined erythema was observed in 1/3 test sites at the 24-, 48-, and 72-hour scoring intervals. The dermal irritation was not observed in 1/3 test sites on Day 7, but was again observed in that animal on Day 10. Dermal irritation did not resolve completely in the remaining 2 test sites. Additional dermal findings included blanching (focal and/or pinpoint areas up to 10 % of the test site) in 1/3 test sites and desquamation in 3/3 test sites.

 

In conclusion, N-Butylpyrrolidone is considered to be a slight irritant to the skin of the rabbit under the conditions of the test (4 hour exposure). The calculated Primary Irritation Index (P.I.I) for the test substance was 1.33.

According to the EEC Dermal Evaluation Criteria, N-Butylpyrrolidone is considered to be a nonirritant for erythema and oedema as listed below:

Animal No.

Erythema

Edema

P.I.I.

3-Minute Exposure

1

0.00

0.00

0.00 - Nonirritant

Irritation Rating

Nonirritant

Nonirritant

1-Hour Exposure

1

1.00

0.00

0.75 - Slight irritant

Irritation Rating

Nonirritant

Nonirritant

4-Hour Exposure

1

2.00

0.00

1.33 - Slight irritant

2

1.00

0.00

3

1.00

0.00

Irritation Rating

Nonirritant

Nonirritant

Eye irritation (in vivo)

The objective of this study was to assess the irritant and/or corrosive effects of N-Butylpyrrolidone when given as a single ocular administration to rabbits (OECD 405; Charles River Laboratories, 2014b; Report No. 20054210). Each of 3 New Zealand White rabbits received a 0.1 mL dose of the test substance in the conjunctival sac of the right eye. The contralateral eye of each animal remained untreated and served as a control. Test and control eyes were examined for signs of irritation for up to 10 days following dosing.

Exposure to the test article produced corneal opacity in 1/3 test eyes by the 24-hour scoring interval and complete resolution occurred by the Day 7 scoring interval. Iritis was observed in 1/3 test eyes by the 1-hour scoring interval and in 2/3 test eyes by the 24-hour scoring interval. Complete resolution of the iritis occurred in 1/3 test eyes by the Day 7 scoring interval and in 2/3 test eyes by the Day 10 scoring interval. Conjunctivitis (redness, swelling, and/or discharge) occurred in 3/3 test eyes by the 1-hour scoring interval and complete resolution occurred in 1/3 test eyes by the Day 7 scoring interval and in 2/3 test eyes by the Day 10 scoring interval. An additional ocular finding of neovascularization (2/3 test eyes) was noted during the study. Decreased fecal output was observed in all animals. This observation, which is a known pharmacological effect of opioids, was likely due to the buprenorphine treatment that the animals received from Days 0 to 5. According to the Kay and Calandra Evaluation Criteria, N-Butylpyrrolidone is considered to be a moderate irritant to the ocular tissue of the rabbit. The total ocular irritation score for the 24-, 48-, and 72-hour intervals was individually added for corneal opacity, iris lesion, conjunctival redness, and conjunctival oedema. Mean scores from gradings at 24, 48 and 72 hours after application of N-Butylpyrrolidone are indicated below:

Animal No./Sex

Corneal Opacity

Iris Lesion

Conjunctival Redness

Conjunctival Edema

5981/M

2.00

1.00

3.00

2.00

5984/M

0.00

1.00

2.67

2.00

5985/M

0.00

1.00

2.00

0.33

Irritation Rating

Nonirritant

Irritant

Irritant

Irritant

According to the EEC Ocular Evaluation Criteria, the substance is classified as non-irritant for corneal opacity but irritant for iritis and conjunctival redness and oedema. According to the CLP criteria, N-Butylpyrrolidone is classified as a Category 2 irritant to the eye (potential to induce reversible eye irritation).

Justification for classification or non-classification

Skin irritation

According to the classification criteria outlined in the section 3.2.2.2. (Guidance on the Application of CLP criteria, 2013), if the mean value of ≥ 2.3 ≤ 4.0 was reached for erythema/eschar or for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal, the classification as Skin Irritant would be assigned. The mean values for erythema and oedema measured in the in vivo study are all under the cut-off values triggering C&L. The mean values for erythema/eschar from gradings at 24 -h, 48 -h and 72 -h after patch removal were 2.0, 1.0 and 1.0 in animal 1, 2 and 3, respectively. The mean values of 0.0 for oedema were measured at the same reading points in each animal.  On the other hand, the irritation responses after 4-hour exposure were delayed (the dermal irritation was not observed in one animal on day 7, but was again observed in that animal on day 10) and persisted to the end of the observation period (day 14) in two animals. The results of in vitro studies have not been taken into account for the purposes of classification and labelling since they are overruled by the in vivo results. Based on this data, the target substance N-Butylpyrrolidone meets criteria under Regulation (EC) No 1272/2008 for classification and labelling as a skin irritant (Cat. 2, H315: Causes skin irritation).

Eye irritation

According to the classification criteria outlined in the section 3.3.2.2. (Guidance on the Application of CLP criteria, 2013), if a substance cause positive but reversible reactions such as corneal opacity (score ≥ 1), iritis (score ≥ 1), conjunctival redness (score ≥ 2) or conjunctival oedema (chemosis) (score ≥ 2) in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after application, the classification as Eye Irritant (Category 2) is assigned. The average irritation scores over 24, 48, and 72 hours for corneal opacity were 2.0, 0.0 and 0.0 in animal 1, 2 and 3, respectively. Iritis mean score of 1.0 was yielded for each of the test animal. The conjunctivae mean scores for redness were 3.0, 2.67 and 2.0 for animal 1, 2 and 3, respectively. The chemosis mean scores of 2.0, 2.0 and 0.33 have been reported for the three tested animals, respectively. According to these values, the positive responders were: one out of three animals for corneal opacity, three animals for iritis and conjunctival redness as well as 2 animals for conjunctival chemosis. Therefore, according to Regulation (EC) No. 1272/2008, N-Butylpyrrolidone should be classified as a Category 2 irritant to the eye (H319: Causes serious eye irritation).