4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Endpoint summary

Description of key information

In vivo guideline studies on irritation are available for rabbit skin and eye. Eye irritation scores met the criteria for classification. Test material residuals prevented appropriate scoring of skin reactions at 24-72hr after application, and the scores would not trigger classification, but based on skin effects such as scaling, baldness and slight erythema observed at 7 -21 days, Mortrace SB will be considered a skin irritant based on non-score considerations. 

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:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was carried according to OECD Guideline No. 404 and EEC Directive 84/449/EEC, Part 8.4 and in accordance with the Principles of Good Laboratory Practices (GLP) as the remanants of the test substance could not be removed from the treatment area and the scores were from approximately 25% of the exposed area.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Broekman Institute, Someren, The Netherlands
- Age at study initiation: approximately 13 weeks
- Weight at study initiation: 2748 - 3096 grams
- Housing: individually housed
- Diet: standard rabbit pelleted diet provided approximately 100 grams/day
- Water: ad libitum
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 grams

Duration of treatment / exposure:

4 hours

Observation period:

The skin reactions were assessed at approximately 45 minutes, 24, 48 and 72 hours and 7, 14 and 21 days after the removal of the dressings and test substance.

Number of animals:

3 male rabbits

Details on study design:

Approximately 24 hours before treatment, the dorsal fur was shaved with electric clippers, exposing an area of approximately 150 square centimeters (10 cm x 15 cm).
Prior to test substance administration, the animals were physically examined and the shaved area of skin to be treated was normal.
On test day 1, 0,5 gram of the test substance was applied to the intact skin of the shaved area on one flank, using a Scotchpak non-woven patch (2x3 cm, 3M, St.Paul, U.S.A.) mounted on Micropore tape (3M, St. Paul, U.S.A.). The contralateral flank was similarly prepared (but without test substance) to act as a procedural control. The dressing was wrapped around the abdomen and secured with an elastic bandage (Coban, 3M, St. Paul, U.S.A.).
Four hours after the application, the dressing was removed and an attempt was made to remove the remaining test substance, using a tissue moistened with tap water and subsequently a dry tissue. However, a brown/yellow sticky layer remained in the treated area.
In order to facilitate the scoring, the skin sites concerned of all animals were reshaved before the observation on days 2, 3, 4, 15 and 22. However, reshaving on days 2, 3 and 4 of the treated skin sites was difficult due to the presence of sticky remnants of’ the test substance.

Irritation parameter:

erythema score

Basis:

animal: rabbit # 3987

Time point:

other: 24, 48 and 72 hours

Score:

0

Reversibility:

other: not applicable

Irritation parameter:

erythema score

Basis:

animal: rabbit # 3988

Time point:

other: 24, 48 and 72 hours

Reversibility:

other: No data

Remarks on result:

not determinable

Remarks:

Erythema could not be scored due to the presence of a brown/yellow, sticky layer of remnants of the test substance in the area of application.

Irritation parameter:

erythema score

Basis:

animal: rabbit # 3989

Time point:

other: 24, 48 and 72 hours

Reversibility:

other: No data

Remarks on result:

not determinable

Remarks:

Erythema could not be scored due to the presence of a brown/yellow, sticky layer of remnants of the test substance in the area of application

Irritation parameter:

edema score

Basis:

animal: rabbit #3987

Time point:

other: 24, 48 and 72 hours

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal: rabbit # 3988

Time point:

other: 24, 48 and 72 hours

Score:

1.3

Max. score:

1

Reversibility:

fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal: rabbit # 3989

Time point:

other: 24, 48 and 72 hours

Score:

1.3

Max. score:

2

Reversibility:

not fully reversible within: 21 days

Irritant / corrosive response data:

The test substance could not be removed from the treated skin sites after the 4 hour exposure. The presence of a brown/yellow, sticky layer made scoring of (large parts of) the skin sites impossible for erythema and/or oedema for the first four days of the study. The parts of’ the skin that could be scored, showed no or very slight erythema and very slight or slight oedema. At the observation 7 days after exposure the treated skin sites of all three animals were noted to be bald and showed moderate to severe erytherna and very slight oedema. The sites were covered with a thick layer of scales, mixed with remnants of the test substance. The skin irritation had resolved within 14 days after exposure in animals 3987 and 3988. Slight skin irritation and scaliness persisted in animal 3989 at the 14-day and 21-day observations. The treated skin of all three animals was still noted to be bald at the final observation.
There was no evidence of a corrosive effect on the skin.

Other effects:

A brown/yellow sticky layer of remnants of the test substance could not be removed from the treated area after the 4 hour exposure period and was also noted at the observations on days 1 to 4 in all three animals. Brown/yellow remnants of the test substance were still noted in the treated area on day 8 in all three animals and on days 15 and 22 in animal 3989.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual scores

24 hours-

Rabbit	Erythema	Edema	Comments
3987	0	2	Approx. 75% of the treated area could not be scored due to the presence of a brown/yellow, sticky layer of remnants of the test substance.
3988	-	1	Oedema could be scored for approx. 25% of the treated area. Scoring of the remaining area was impossible due to the presence of remnants of the test substance.
3899	-	1	same as above

- Erythema could not be scored due to the presence of a brown/yellow, sticky layer of remnants of the test substance in the area of’ application.

48- hours-

Rabbit	Erythema	Edema	Comments
3987	0	1	Approx. 75% of the treated area could not be scored due to the presence of a brown/yellow, sticky layer of remnants of the test substance.
3988	-	1	In animals 3988 and 3989, oedema could be scored forapprox. 25% of the treated area. Scoring of the remaining area was impossible due to the presence of remnants of the test substance.
3899	-	1	same as above

- Erythema could not be scored due to the presence of a brown/yellow, sticky layer of remnants of the test substance in the area of’ application.

72 -hours-

Rabbit	Erythema	Edema	Comments
3987	0	1	In all three animals, both erythenia and oederna could be scored for approx. 25% of the treated area. Scoring of the remaining area was impossible due to the presence or a brown/yellow, sticky layer of remnants of the test substance.
3988	1	2	same as above
3899	1	2	same as above

- Erythema could not be scored due to the presence of a brown/yellow, sticky layer of remnants of the test substance in the area of’ application.

7 days-

Rabbit	Erythema	Edema	Comments
3987	3	1	In all three animals a thick layer of scales, mixed with brown/yellow remnants of the test substance, was seen in the area of application. Underneath, a bald skin with erythema score 3 was observed.
3988	3	1	same as above
3899	3	1	same as above

14 days-

Rabbit	Erythema	Edema	Comments
3987	0	0	In all three animals, approx. 80% of’ the treated skin was noted to be bald. Scaliness, mixed with orange/yellow remnants of the test substance, was noted in approx. 50% of the treated area of animal 3989.
3988	0	0	same as above
3899	0	1	same as above

21 days-

Rabbit	Erythema	Edema	Comments
3987	0	0	In all three animals, approx. 80% of the treated skin was noted to be bald. In animal 3989, the bald skin showed erythema score 1 and scaliness, mixed with orange/yellow remnants of’ the test substance, was noted at the edges of the treated area.
3988	0	0	same as above
3899	1	2	same as above

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Using the available scores a primary irritation index of 1.8 was calculated, indicating that MORTRACE SB CONC. was mildly irritating to the rabbit skin. However, the appearance of a bald skin, moderate to severe erythema and scaliness 7 days after exposure indicate that the scores obtained at the 24 to 72 hour observations were probably an underestimation of the irritating potential. Therefore, for the purpose of safety evaluation, NORTRACE SB CDNC. was considered moderately irritating.

Executive summary:

The purpose of this study was to assess the possible skin irritation or skin corrosion potential of MORTRACE SB CONC. in rabbits.

This study was carried out in accordance with DECO Guideline No. 404, “Acute Dermal Irritation / Corrosion” and EEC Directive 84/449/EEC, Part 8.4, “Acute Toxicity - Skin Irritation”.

MORTRACE SB CONC. was applied onto shaved skin of three male albino rabbits using semi-occlusive dressings for 4 hours, followed by seven observations at approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after removal of the dressings and remaining test substance.

The test substance could not be removed from the treated skin sites and made scoring impossible and/or difficult during the first four days of the study. The parts of the skin that could be scored, showed only slight irritation. 7 Days after exposure a bald skin, scaliness and moderate to severe erythema and very slight oedema were noted in all three animals. The skin irritation had resolved within 14 days after exposure in two animals and slight skin irritation and scaliness persisted in one animal untill termination (day 22). There was no evidence of a corrosive effect on the skin.

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

A primary irritation index of 1.8, calculated using the available scores, indicate that MORTRACE SB CONe, was mildly irritating to the rabbit skin. However, based on the clinical signs noted from day 7 onwards the test substance was considered moderately irritating, for the purpose of safety evaluation.

Due to the absence of critical scores, classification of MORTRACE SB CONC. in accordance to the EEC criteria for general classification and labelling requirements for dangerous substances and preparations (EEC Directive 91/325/EEC, Amendment to Annex VI of the EEC Directive 67/548/EEC) was difficult. The clinical signs observed from day 7 onwards indicated an irritating potential of the test substance when applied to the rabbit skin. Therefore it was considered that MORTRACE SB CONC. should be labelled as a skin irritant.

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:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 405 and EEC Directive 84/449/EEC Part B.5 and in accordance with the Principles of Good Laboratory Practices (GLP)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Broekman Institute, Someren, The Netherlands
- Age at study initiation: approximately 14-18 weeks
- Weight at study initiation: 3003 grams - 3325 grams
- Housing: individually housed
- Diet: standard rabbit pelleted diet provided approximately 100 grams/day
- Water: ad libitum
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

100 ± 2 mg/animal

Duration of treatment / exposure:

as per guideline

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 and 14 days after instillation of the test substance.

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

Prior to test substance administration, each of the animals were physically examined and the eyes were found to be normal.
The test substance (100 ± 0.2 mg per animal) was instilled in the conjunctival sac of the left eye of one animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about ten seconds to prevent loss of the test substance. The other eye remained untreated and served as the reference control
Immediately after 24 hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively
determine corneal epithelial damage. This procedure was repeated in on day 4, until recovery was completed. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
The two other animals were treated in a similar manner one week later, after considering the degree of eye irritation observed in the first animal. Treatment of the eyes with a fluorescein solution was performed immediately after the observations on days 2, 4 and/or day 8 in these animals.

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal: rabbit 3972

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal: rabbit 3979

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal: rabbit 4011

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

iris score

Remarks:

mean

Basis:

animal: rabbit 3972

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

iris score

Remarks:

mean

Basis:

animal: rabbit 3979

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

iris score

Remarks:

mean

Basis:

animal: rabbit 4011

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal: rabbit 3972

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal: rabbit 3979

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal: rabbit 4011

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Remarks:

mean

Basis:

animal: rabbit 3972

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Remarks:

mean

Basis:

animal: rabbit 3979

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Remarks:

mean

Basis:

animal: rabbit 4011

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

One hour after instillation of MORTRACE SB CONC. into one eye of each of the three male albino rabbits resulted in slight redness and moderate to severe swelling in the conjunctivae. Increase of the conjunctival redness was noted at the 24-hour observation, whereas iridic irritation and slight dulling of normal lustre in the cornea (in two animals only) were also observed. Corneal epithelial damage, determined by fluorescein, was seen on day 2 in all three animals and persisted on day 4 in animal 4011. The corneal damage and iridic irritation had resolved within 48 hours after instillation in animals 3972 and 3979 and within 7 days in animal 4011. The irritation of the conjunctivae had resolved within 14 days in all three animals.
There was no evidence of ocular corrosion.

Other effects:

The outside of the eyelids were covered with a brown, sticky layer of test substance during the study period. The layer had disappeared at the 14-day observation and a yellow staining of the eyelids remained. No toxic symptoms were observed in the animals during the test period and no mortality occurred.

Individual Scores

Cornea opacity
Animal	Observation time			Mean value	Mean value (over all animals)
	24 hours	48 hours	72 hours
3972	0	0	0	0.0	0.0
3979	0	0	0	0.0
4011	0	0	0	0.0
Iris lesion
Animal	Observation time			Mean value	Mean value (over all animals)
	24 hours	48 hours	72 hours
3972	1	0	0	0.3	0.6
3979	1	0	0	0.3
4011	1	1	1	1.0
Conjunctivae (redness)
Animal	Observation time			Mean value	Mean value (over all animals)
	24 hours	48 hours	72 hours
3972	3	3	2	2.7	2.8
3979	3	3	3	3.0
4011	2	3	3	2.7
Conjunctivae (chemosis)
Animal	Observation time			Mean value	Mean value (over all animals)
	24 hours	48 hours	72 hours
3972	1	1	1	1.0	1.2
3979	2	1	1	1.3
4011	2	1	1	1.3

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

MORTRACE SB CONC. was considered mildly irritating (M2) to the rabbit eye (Kay and Calandra interpretation of the calculated maximum Draize score: 12.3). According to the EEC criteria for general classification and labelling requirements for dangerous substances and preparations (EEC Directive 91/325/EEC, Amendment to Annex VI of the Directive 67/548/EEC), MORTRACE SB CONC. should be labelled as an eye irritant.

Executive summary:

The purpose of this study was to assess the possible irritation or corrosion potential when a single dose of MORTRACE SB CONC. was placed in the conjunctival sac of a rabbit eye.

This study was carried out in accordance with OECD Guideline No. 405, “Acute Eye Irritation / Corrosion” and EEC Directive 84/449/EEC, Part 8.5, “Acute Toxicity — Eye Irritation”.

MORTRACE SB CONC. was instilled into one eye of each of three male albino rabbits, followed by six observations at approximately 1, 24, 48 and 72 hours and 7 and 14 days after test substance administration.

Under the conditions of this study, MORTRACE SB CONC. resulted in adverse effects on the cornea, iris and the conjunctivae. Corrieal epithelial damage was observed in all three animals, which resulted in slight dulling of normal lustre in two animals. The corneal damage had resolved within 2 or 7 days. The iridic irritation had resolved within 2-7 days and the irritation of the conjunctivae had resolved within 14 days after instillation in the animals. Ocular corrosion was not observed in any of the rabbits.

Brown, sticky remnants of and yellow staining by the test substance were noted on the outside of the eyelids during the study period.

No signs of systemic intoxication were observed during the study period.

MORTRACE SB CONC. was considered mildly irritating (N2) to the rabbit eye (Kay and Calancira interpretation of the calculated maximum Draize score: 12.3). According to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (EEC Directive 91/325/EEC, Amendment to Annex VI of the EEC Council Directive 67/548/EEC), NORTRACE SB CONC. should be labelled as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD Guideline GLP studies are available for both skin and eye irritation.

In the key study for skin irritation although the scores for erythema and edema would not support the conclusion of irritating, the test material produced scaling of the skin and baldness around the site of application that persisted to the end of the test period, leading to the conclusion that this substance is meets the criteria for classification as an irritant.

In the Eye irritation assay, application of Mortrace SB CONC to the eyes of rabbits resulted in adverse effects on the cornea, iris and the conjunctivae. Corrieal epithelial damage was observed in all three animals, which resulted in slight dulling of normal lustre in two animals. The corneal damage had resolved within 2 or 7 days. The iridic irritation had resolved within 2-7 days and the irritation of the conjunctivae had resolved within 14 days after instillation in the animals. Occular corrosion was not observed in any of the rabbits.

Brown, sticky remnants of and yellow staining by the test substance were noted on the outside of the eyelids during the study period.

No signs of systemic intoxication were observed during the study period.

Based on the observed irritation, Mortrace SB is consdiered to be irritating to the eyes.

Justification for selection of skin irritation / corrosion endpoint:
GLP guideline skin irritation study

Justification for selection of eye irritation endpoint:
GLP guideline eye irritation study

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

In vivo guideline studies on irritation are available for rabbit skin and eye. Eye irritation scores met the CLP criteria for classification as eye irritant. Test material residuals prevented appropriate scoring of skin reactions at 24-72hr after application, and the scores would not trigger classification, but based on skin effects such as scaling, baldness and slight erythema observed at 7 -21 days, Mortrace SB will be considered a skin irritant based on non-score considerations.