4'-tert-butyl-2',6'-dimethyl-3',5'-dinitroacetophenone

Endpoint summary

Description of key information

Skin irritation/corrosion: Not irritating (OECD 439/GLP)

Serious eye damage/eye irritation (in vivo): Not Irritating (OECD 405/GLP)

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:

13-11-2017 to 19-12-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Puyang Ouya Chemical Technology Co., Ltd; 2017073046
- Expiration date of the lot/batch: 31st December 2018
- Purity: 99.87 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light and water

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SK) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media. The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia): Lot No. 25860 kit A. The certificate of analysis is presented in Annex 1

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 minutes at room temperature and the remaining 35 minutes at 37±1°C
- Temperature of post-treatment incubation (if applicable): 42 hours at 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Tissues were then thoroughly rinsed with PBS
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: No deviations

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT (1mg/mL)
- Spectrophotometer: Libra S22
- Wavelength: 570 nm
- Filter: No external filter was used
- Filter bandwidth: Allowed band width is 2-3 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Triplicates for test substance, positive and negative controls.

VIABILITY CALCULATION
Relative cell viability was calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test item was calculated – this value is used for the comparison with limit.

PREDICTION MODEL / DECISION CRITERIA
Evaluation of results and classification
In vitro alternatives that have been validated and accepted may also be used to help in classification decisions making (see Regulation (EC) 1272/2008, 3.2. Skin corrosion/ irritation, 3.2.2. Classification criteria for substances). For further classification, the relative cell viability is calculated for each tissue as % of the mean of the negative control tissues viability, which is set at 100 %.
The cut-off values for the prediction of irritation are given below; these values are stated in OECD Test Guideline No. 439, par. 36:

-In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS * Category 2.
-The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: Direct MTT reduction control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at 37±1°C)

Duration of post-treatment incubation (if applicable):

42 hours at 37±1°C

Number of replicates:

A single experiment, composed of three replicate tissues for each treatment group, was run.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Musk Ketone

Value:

102.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SD:1.2%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

PBS

Value:

100

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SD:3%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

5% SDS

Value:

2.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SD:0.2%

Other effects / acceptance of results:

- Visible damage on test system: No changes in tissue appearance were observed during the experiment.

- Direct-MTT reduction: After approximately 1 hour incubation of the test item with MTT medium at culture conditions, the MTT medium did not change its colour (See Figure 1). The test item did not reduce MTT directly so other steps did not have been performed.

- Colour interference with MTT: The result of measuring of OD570 extract from the test item on the 96-well plate was the following:

OD570 of isopropyl alcohol (average from 2 wells): 0.052
OD570 of the test item (average from 2 wells): 0.043.
Net OD570: 0.043-0.052= -0.009

As the net OD570 for the test item was < 0.08, colour of the test item did not interfere with the endpoint, so next steps did not need to be performed.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 of the negative control tissue was 1.893 which meets the acceptance criteria of ≥ 0.8 and≤ 2.8.

- Acceptance criteria met for positive control: The mean viability of the positive control tissues expressed as % of the negative control tissues was 2.4 % which meets the acceptance criterion of ≤ 20 %.

- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual % tissue viabilities of the 3 identically treated replicates was 0.2 % for the positive control, 3.0 % for negative control and 1.2 % for the test item what is < 18 % in all cases.

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro skin irritation test (EpiDermTM Model), Musk Ketone was not irritating.

Executive summary:

In an in vitro skin irritation assay in a human epidermal model EpiDerm (17-767), PBS-moistened reconstructed human epidermis tissue was exposed to 25 mg of musk ketone (99.87%) for 60±1 minutes (25 minutes at room temperature and 35 minutes at 37±1°C). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 135 minute isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance musk ketone was 102.4 % ±1.2% of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 2.4 % ±0.2% of negative control average value. According to these results, the test substance is not irritating.

This in vitro skin irritation study in the human epidermal model EpiDerm is acceptable and satisfies the guideline requirement for an OECD 439 study.

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:

12-09-1996 to 21-02-1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RIFM; 9615500072

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: ground in a mortar with a pestle, and passed through a No. 40 mesh sieve

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Myrtle’s rabbitry, TN, USA
- Age at study initiation: adult
- Weight at study initiation:2.8 kg
- Housing: Individually in stainless stain cages
- Diet: PMI certified rabbit chow #5322 (Purina Mills Inc) ad libitum
- Water: Municipal tap water treated by reverse osmosis ad libitum
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18.2-20.5°C
- Humidity: 26-31%
- Air changes (per hr): 10-15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):70 mg

Duration of treatment / exposure:

Rinsed group: 30 seconds
Non-rinsed group:72 hours

Observation period (in vivo):

Rinsed and non-rinsed groups: 1, 24, 48 and 72 hours

Number of animals or in vitro replicates:

Rinsed group: 3 females
Non-rinsed group: 2 males and 1 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes (rinsed group)
- Time after start of exposure: Approximately 30 seconds after instillation of the test article, with physiological saline.

SCORING SYSTEM & TOOL USED TO ASSESS SCORE: The eyes were macroscopically examined with the aid of an auxillary light source for signs of irritation at 1, 24, 48 and 72 hours after dosing according to the Ocular Grading System presented in Protocol Appendix A 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 article was gently rinsed from the eye at this time (if possible) using physiological saline. If any 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.

Irritation parameter:

cornea opacity score

Remarks:

No rinse group

Basis:

animal: 1, 2, 3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Remarks:

No rinse group

Basis:

animal: 1, 2, 3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Remarks:

No rinse group

Basis:

animal #1

Time point:

24 h

Score:

4

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

No rinse group

Basis:

animal #1

Time point:

other: 48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Remarks:

No rinse group

Basis:

animal #2

Time point:

24 h

Score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

No rinse group

Basis:

animal #2

Time point:

other: 48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Remarks:

No rinse group

Basis:

animal #3

Time point:

24 h

Score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

No rinse group

Basis:

animal #3

Time point:

other: 48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Remarks:

Rinse group

Basis:

animal: 1, 2, 3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Remarks:

Rinse group

Basis:

animal: 1, 2, 3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Remarks:

Rinse group

Basis:

animal: 1, 2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Remarks:

Rinse group

Basis:

animal #3

Time point:

24/48 h

Score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

Rinse group

Basis:

animal #3

Time point:

72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal: 1, 2, 3

Time point:

24/48/72 h

Score:

0

Remarks on result:

other:

Remarks:

Included in conjunctival score

Irritant / corrosive response data:

Iris scores = Iritis X 5
Cornea scores = Opacity x Area x 5
Conjunctival scores = (Redness + Swelling + Discharge) X 2

No rinse group: Exposure to the test article produced conjunctivitis (redness, swelling and/or discharge) in the 3/3 test eyes at the 1 hour scoring interval (Table 1). The conjunctival irritation resolved completely in all animals by the 48 hours scoring interval. No corneal opacity or iritis was observed in the test eyes at any time-point. No corneal opacity, iritis or conjunctivitis was observed in the control eyes.

Rinsed group: Exposure to the test article produced conjunctivitis (redness and swelling) in 3/3 test eyes at the 1 hour scoring interval (Table 2). The conjunctival irritation resolved completely in all animals by the 72 hour scoring interval. No corneal opacity or iritis was observed in the test eyes at any time-point. No corneal opacity, iritis or conjunctivitis was observed in the control eyes.

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo eye irritation study in rabbits, musk ketone is not irritating.

Executive summary:

In a primary eye irritation study (29495), 70 mg of musk ketone (undiluted) was instilled into the conjunctival sac of the right eye of adult New Zealand rabbits (3 females; rinsed group and 2 males and 1 female; non-rinsed group). Approximately 30 seconds after instillation of the test article, eyes of the rinsed group were washed with physiological saline. The remaining rabbits were not rinsed. Animals then were observed for 3 days.  Irritation was scored on a method based on Draize.

In the group of animals with no rinsing, exposure to the test article produced conjunctivitis (redness, swelling and/or discharge) in the 3/3 test eyes at the 1 hour scoring interval . The conjunctival irritation resolved completely in all animals by the 48 hours scoring interval. No corneal opacity or iritis was observed in the test eyes at any time-point. No corneal opacity, iritis or conjunctivitis was observed in the control eyes. In the group of animals with rinsing, exposure to the test article produced conjunctivitis (redness and swelling) in 3/3 test eyes at the 1 hour scoring interval. The conjunctival irritation resolved completely in all animals by the 72 hour scoring interval. No corneal opacity or iritis was observed in the test eyes at any time-point. No corneal opacity, iritis or conjunctivitis was observed in the control eyes.

In this study, musk ketone is not an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

There is one in vitro skin irritation study available.

In an in vitro skin irritation assay in a human epidermal model EpiDerm (OECD 439/GLP), PBS-moistened reconstructed human epidermis tissue was exposed to 25 mg of musk ketone (99.87%) for 60±1 minutes (25 minutes at room temperature and 35 minutes at 37±1°C). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 135 minute isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

 

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance musk ketone was 102.4 % ±1.2% of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 2.4 % ±0.2% of negative control average value. According to these results, the test substance is not irritating.

Serious eye damage/eye irritation (in vivo):

There is no in vitro data available and there is one in vivo primary eye irritation study available.

 

In a primary eye irritation study (OECD 405/GLP), 70 mg of musk ketone (undiluted) was instilled into the conjunctival sac of the right eye of adult New Zealand rabbits (3 females; rinsed group and 2 males and 1 female; non-rinsed group). Approximately 30 seconds after instillation of the test article, eyes of the rinsed group were washed with physiological saline. The remaining rabbits were not rinsed. Animals then were observed for 3 days.  Irritation was scored on a method based on Draize.

 

In the group of animals with no rinsing, exposure to the test article produced conjunctivitis (redness, swelling and/or discharge) in the 3/3 test eyes at the 1 hour scoring interval . The conjunctival irritation resolved completely in all animals by the 48 hours scoring interval. No corneal opacity or iritis was observed in the test eyes at any time-point. No corneal opacity, iritis or conjunctivitis was observed in the control eyes. In the group of animals with rinsing, exposure to the test article produced conjunctivitis (redness and swelling) in 3/3 test eyes at the 1 hour scoring interval. The conjunctival irritation resolved completely in all animals by the 72 hour scoring interval. No corneal opacity or iritis was observed in the test eyes at any time-point. No corneal opacity, iritis or conjunctivitis was observed in the control eyes. In this study, musk ketone is not an eye irritant.

Both studies are suitable to use in the human health hazard assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance musk ketone (CAS No. 81-14-1) does not need to be classified for skin irritation/corrosion  or serious eye damage/eye irritation when considering the criteria outlined in Annex I of 1272/2008/EC.