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Administrative data

Description of key information

An in vitro skin irritation study is available according to OECD TG 439 is available and BMI was shown to be a non-irritant.

An in vitro eye study study (OECD TG 437) performed with BMI was inconclusive. Subsequently an in vivo eye irritation study was performed (OECD TG 405). Minimal irritation was observed and BMI was concluded not to be an eye-irritant.

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
Study period:
From 16 May to 18 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Epidermal cells from adult donors
Justification for test system used:
One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small Model
- Tissue batch number: 12-EKIN-024
- Surface: 0.38 cm^2
- Expiration date: 18 June 2012

CHECK FOR REDUCTION OF MTT BY TEST SUBSTANCE
- The MTT reducing capability of the test substance was investigated by mixing approximately 10 mg of the test substance with 2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 μL of distilled water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

CHECK FOR COLOR INTERFERENCE OF MTT BY TEST SUBSTANCE
- The test substance was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 10.1 mg of the test substance with 91 μL of distilled water in a transparent container to give 10 mg/90 μL. 100 μL of distilled water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the colour of the solution was assessed by eye.

CHECK FOR PH
An estimate of the pH of the test substance as a 10% suspension in distilled water was determined using pH indicator paper and recorded.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation (if applicable): 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air + 3 hours ± 5 minutes with MTT .

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT :
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours ± 5 minutes at 37 ± 2°C
- Wavelength of OD measurements: 540 nm

VIABILITY MEASUREMENTS PREPARATION
AFTER all the tissues had been punched, the tissues (epidermis and collagen matrix) were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).
The tissues were extracted by storing at 2-8 ºC, protected from light, for 48 - 70 hours. After formazan extraction, duplicate 200 μL 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.

NUMBER OF REPLICATE TISSUES: triplicate tissues with the test item, negative and positive control.

PREDICTION MODEL / DECISION CRITERIA
If the mean tissue viability was equal to or less than 50% of the negative control value, the sample was classed as Irritant R38 (EU classification) or Category 2 (GHS classification).

ACCEPTANCE CRITERIA:
The OD from the negative control tissue in the MTT assay is an indicator of tissue viability after the shipping and storage procedure and under the specific conditions of the assay. The mean absorbance of the triplicate negative control values should be ≥0.6 and ≤1.5. The Standard Deviation (SD) value of the % viability should be <18.

The OD of the positive control is an indicator of the sensitivity of the tissues. The mean viability should be ≤40% of the negative control and the SD <18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 ± 2 mg (spread over the surface of the tissue using a curved spatula)
- The tissues were wetted with 5 μL of distilled water prior to application of the test material.
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hour ± 1 hours ; + 3 hours ± 5 minutes with MTT
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 experiments
Value:
91.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Negative control
The mean absorbance of the triplicate negative control values was 0.917 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 5.9 which was below the maximum value of 18.
Positive control
The percentage mean viability of the positive control was 10.9 ± 2.9 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

Results of individual reports are reported in "any other information"

The results of the assay are summarised in the table below.

Sample

Tissue viability as percentage of mean OD negative control

Prediction MTT endpoint

Replicate Tissues

Mean ± SD

a

b

c

Negative control

99.2

106.3

94.6

100.0 ± 5.9

Not applicable

Positive control

12.9

12.3

7.6

10.9 ± 2.9

Irritant

Test substance

96.8

92.0

86.3

91.7 ± 5.3

Not-irritant

 

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles, BMI showed to be a non-irritant (tissue viability of 91.7± 5.3%). Therefore, the substance was predicted as non-irritant to the skin.
Executive summary:

In an in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles. The influence of BMI on the viability of human skin was tested. Ca. 10 mg of test substance was applied directly on top of 0.38 cm2cultured skin. After 15 minutes, the substance was removed and cells were cultured for 42 hours. BMI showed to be a non-irritant (mean tissue viability of 91.7%). Therefore, the substance does not need to be classified according to regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 August 2012 - 07 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
April 2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Eye Irritation (2-1-5), 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 31 or 39 weeks
- Weight at study initiation: 3.69 to 4.46 kg
- Housing: Each animal was housed individually in a plastic cage with perforated floors
- Diet (e.g. ad libitum): standard laboratory rabbit diet per day
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 11 or 17 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16- 20
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

Vehicle:
not specified
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL


Duration of treatment / exposure:
After instillation of the test substance the eyelids of each animal were gently held together for one second before releasing
Observation period (in vivo):
1, 24,48, 72 and 8 days after instillation
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Eyes were flushed with saline for animal #1 24 hours after instillation, Eyes were flushed with saline for animal #2 24 hours and 48 hours after instillation. Eyes were not flushed for animal #3

SCORING SYSTEM: Kay and Calandra and EC (Regulation 1272/2008) and GHS classification



Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
Animal #1;#2;#3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: n/a
Remarks on result:
other: The scores are calculated at approximately 24, 48 and 72 hours after administration, separately for each animal
Irritation parameter:
iris score
Basis:
mean
Remarks:
Animal #1;#2;#3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: n/a
Remarks on result:
other: The scores are calculated at approximately 24, 48 and 72 hours after administration, separately for each animal
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 8 days
Remarks on result:
other: The scores are calculated at approximately 24, 48 and 72 hours after administration, separately for each animal
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: The scores are calculated at approximately 24, 48 and 72 hours after administration, separately for each animal
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: The scores are calculated at approximately 24, 48 and 72 hours after administration, separately for each animal
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: The scores are calculated at approximately 24, 48 and 72 hours after administration, separately for each animal
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: The scores are calculated at approximately 24, 48 and 72 hours after administration, separately for each animal
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: n/a
Remarks on result:
other: The scores are calculated at approximately 24, 48 and 72 hours after administration, separately for each animal
Interpretation of results:
GHS criteria not met
Conclusions:
An in vivo eye irritation test was performed in new zealand white rabbits. Based on the results, BMI does not have to be classified for eye irritation or serious eye damage according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Executive summary:

An in vivo eye irritation test was performed in new zealand white rabbits according to internationally accepted guidelines and in accordance with GLP principles. Following a instillation dose of 0.1 mL,

slight chemosis, slight to moderate discharge, and injection of the conjunctival blood vessels were noted in 1 or more animals, all clinical signs were reversed to normal within 8 days in all test animals.

Based on the results, BMI does not have to be classified for eye irritation or serious eye damage according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION

An in vitro skin irritation test, performed according to OECD TG 439 and in accordance with GLP principles. The influence of BMI on the viability of human skin was tested. Ca. 10 mg of test substance was applied directly on top of 0.38 cm2 cultured skin. After 15 minutes, the substance was removed and cells were cultured for 42 hours. BMI showed to be a non-irritant (mean tissue viability of 91.7%).

 

EYE IRRITATION

A Bovine Corneal Opacity and Permeability Assay (BCOP) (in vitro) was undertaken according to OECD TG 437 and in accordance with GLP principles. An In Vitro Irritancy Score (IVIS) was calculated using the following formula: In Vitro Irritancy Score = Corrected Opacity Value + (15 x Corrected OD490Value). The IVIS was calculated for each individual treatment and positive control cornea. The mean IVIS value for each treatment group was calculated from the IVIS of each individual cornea in the treatment group. For BMI, the IVIS score was 6.0± 0.8. Therefore BMI was predicted not to be a corrosive or severe eye irritant and additional testing should be performed for classification and labelling purposes. With the most recent update of OECD TG 437 (2017) the IVIS score of 6.0 would be interpreted as inconclusive.

An in vivo eye irritation test was performed in new zealand white rabbits according to internationally accepted guidelines (OECD TG 405) and in accordance with GLP principles. Following a instillation dose of 0.1 mL, slight chemosis, slight to moderate discharge, and injection of the conjunctival blood vessels were noted in 1 or more animals, all clinical signs were fully reversible within 8 days in all test animals. Based on the result BMI was concluded not to be an eye-irritant.

 

Justification for selection of skin irritation / corrosion endpoint:

The EPISKIN study (OECD TG 437) carried out by Huntingdon Life Sciences was carried out in accordance with GLP and to international guidelines and is thus reliable and is designated the key study.

 

Justification for selection of eye irritation endpoint:

The in vivo eye irritation study (OECD TG 405) carried out by Huntingdon Life Sciences was carried out in accordance with GLP and to international guidelines and is thus reliable and is designated the key study for this endpoint.

Justification for classification or non-classification

SKIN IRRITATION/CORROSION

Based on the results of the in vitro skin irritation test (OECD TG 439) BMI does not warrant classification for skin irritation according to regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

EYE IRRITATION

Based on the results of an in vivo eye irritation study (OECD TG 405) BMI does not warrant classification for eye irritation according to regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

RESPIRATORY IRRITATION

No information is available to enable classification as a respiratory irritant.