Registration Dossier

Administrative data

Description of key information

There is no study available however the registrant has reported cases of workers with throat irritations when breathing NTO dust.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP test performed according to OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Lot number: DDP12K0091-0002
Expiry: 31 December 2022
Purity: 100%
Species:
human
Type of coverage:
other: EPISKIN™ human epidermis skin constructs
Preparation of test site:
other: incubation of at least 24 hours in maintenance medium
Vehicle:
unchanged (no vehicle)
Remarks:
The tissues were wetted with 5 μL of distilled water prior to application of the test substance
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied :10 +/- 2mg of the test substance NTO


Duration of treatment / exposure:
15 minutes treatment
Details on study design:
After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 +/- 0.5 minutes with the test substance, negative or positive control at room temperature. One additional tissue was dosed with the test substance, NTO, and another tissue dosed with DPBS for the colour controls. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30seconds and then re-spread with a curved flat spatula after 7 minutes application time.
After 15 +/-0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbecco’s Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 +/- 1 hour at 37 +/- 2oC in a humidified atmosphere of 5% CO2 in air. After 42 +/- 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours +/- 5 minutes at 37 +/- 2oC in a humidified atmosphere of 5% CO2 in air. The colour controls were transferred to wells containing 2 mL of assay medium without MTT. At the end of 3 hours +/- 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube. When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted for four hours, protected from light. During the four hour extraction, extracts were transported at ambient temperature within the range 19.1 to 20oC, from ERC, Eye, to HRC, Huntingdon. After two hours extraction, at HRC, the extracts were vortexed. After formazan extraction, duplicate 200 uL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.
The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.
Irritation / corrosion parameter:
other:
Value:
ca. 100
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: no data. Remarks: Conclusion: non-irritant to the skin. (migrated information)
Other effects:
There was no change in the colour of the water control after the 15 minute shaking period. The test substance, NTO/water solution was colourless prior to incubation but was pale yellow in colour after incubation. As the test substance had shown colouring of the water, (potential interference with assessment of optical density) colour control tissues were run in parallel with the main tissues but not exposed to MTT.

There was no change in the test substance, NTO/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 +/- 2oC in a humidified atmosphere of 5% CO2 in air. The test substance had not interacted with the MTT.

Negative control: the mean absorbance of the triplicate negative control values was 0.916 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 7.6 which was below the maximum value of 18.

Positive control: The percentage mean viability of the positive control was 14.6 ± 4.2 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

EPISKIN™ results: The percentage value for the OD of test substance NTO, linked to colour was 2.074% of the negative control with MTT (Table 1). As this value was below 5%, correction of the test substance OD values was not required.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The test substance, NTO, elicited a mean tissue viability of 100.3 ± 2.8% using the EPISKIN™ human epidermis skin constructs and therefore was predicted as non-irritant to the skin.
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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed to a reliable method but not to GLP
Qualifier:
according to
Guideline:
other: Guideline used: Interagency Regulatory Liaison Group, "Recommended Guidelines for Acute Eye Irritation Testing," U.S. Consumer Product Safety Commission, Washington, DC, 20207, January 1981.
Principles of method if other than guideline:
Six New Zealand rabbits were used in this facet of the study. The pure compound was instilled in 100 mg quantities into the conjunctival envelope of the left eye of each rabbit; the right eye served as a control . Two of the rabbits had the compound washed from the eye 30 seconds after instillation; two, 5 min after instillation ; and two did not have the compound washed from the eye. Each eye was graded for ocular lesions at 1 and 4 h on the day of the application and again at 24, 48, and 72 h.
GLP compliance:
not specified
Specific details on test material used for the study:
Batch No.: not specified
Purity: not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.4-2.8 kg

No additional data
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg

No additional data
Duration of treatment / exposure:
72 hours
Observation period (in vivo):
1, 4, 24, 48, 72 hours
Number of animals or in vitro replicates:
6
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Two of the rabbits had the compound washed from the eye 30 seconds after instillation; two, 5 min after instillation; and two did not have the compound washed from the eye.

SCORING SYSTEM: Draize

No additional data
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Wash at 30 sec
Irritation parameter:
iris score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Wash at 30 sec
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Wash at 30 sec
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Wash at 5 min
Irritation parameter:
iris score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
1.25
Max. score:
2.5
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: Wash at 5 min
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
2.83
Max. score:
8
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: Wash at 5 min
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: No wash
Irritation parameter:
iris score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: No wash
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: mean of 24, 48 and 72 hours
Score:
1.17
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: No wash
Irritant / corrosive response data:
All rabbit eyes showed erythemia or redness (vascular congestion) of conjunctival vessels for the 1 and 4 h observations. One rabbit eye, from the 5 min "wash" group, had slight cornea opacity after 1 h and severe chemosis at 4 h preventing observation until 24 h. Then the cornea appeared clear, while the iris showed congestion with the pupil reacting slowly t o light. This condition continued through the test period with redness, chemosis, and discharge. The iris congestion was still present after 7 days. After 18 days, the rabbit was killed and tissues were taken for histological studies. When examined microscopically, the eye from the animal had a chronic granulomatous iritis with prominent infiltrations of large mononuclear inflammatory cells.
This iris response is considered a positive reaction but 1 positive response from 6 rabbits is considered a negative result for eye irritation studies according to classical guidelines. After 48 h, one of the remaining rabbits showed slight chemosis which was negative after 72 h.
Other effects:
No information provided

Eye Irritation Responses in Rabbits Treated with Test substancea

Tissue Gradedb

Time to Score

Time to Score

1 hours

2 hours

1 days

2 days

3 days

Wash at 30 sec

Cornea

Iris

Conjunctive

 

0

0

2.0

 

0

0

4.0

 

0

0

0

 

0

0

0

 

0

0

0

Wash at 5 min

Cornea

Iris

Conjunctive

 

12.5

0

8.0

 

*

*

11.0

 

0

2.5

8.0

 

0

2.5

5.0

 

0

2.5

4.0

No Wash

Cornea

Iris

Conjunctive

 

0

0

7.0

 

0

0

7.0

 

0

0

4.0

 

0

0

2.0

 

0

0

1.0

aTwo rabbits per wash conditions.

bMaximum cornea response = 80; maximum iris response = 10 ; maximum conjunctivae response = 20.

*Unable to score cornea and/or iris for swelling

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
With the scoring scheme, the rabbit eye test was considered negative; however, transient conjunctival and corneal irritation did result from the test substance application in several animals and one developed a chronic anterior uveitis.
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:

One reliable GLP in vitro study following international guidelines

Justification for selection of eye irritation endpoint:

One in vivo study available

Justification for classification or non-classification

NTO did not cause any irritation in an in vitro EpinSkin study or an acute dermal study. However, there are reports from the registrant of workers with skin irritation due to NTO exposure on wet unprotected skin and throat irritation due to breathing in NTO dust. Therefore NTO is classified as a skin and eye irritant category 2 and acute inhalation Category 3 in accordance with Regulation (EC) No 1272/2008.