Benzenepropanal, 2-methyl-4-(2-methylpropyl)-

Endpoint summary

Description of key information

Skin irritation:

In vitro skin irritation test with the substance itself was assessed using a human skin model according the OECD Guideline No.: 439.

It was found to be irritant to the skin in vitro.

Eye Irritation:

The substance was found to be eye irritant in an in vivo study in rabbits (OECD 405, GLP), but non eye irritant in an in vivo BCOP test (OECD 437, GLP).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Mar 2015 to 22 May 2015

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)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name (as stated in the report): GR-88-0778
Lot No: Batch 6
Aspect: Colourless liquid
Expiration date: March 29, 2017

Test system:

human skin model

Source species:

human

Cell type:

other: human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM), 0.38 cm2 , Batch no.: 15-EKIN-020)

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

not specified

Details on animal used as source of test system:

adult human-derived epidermal keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. 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)

Remarks:

The liquid test substance was applied undiluted (25 μl) directly on top of the tissue.

Details on test system:

- Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 15-EKIN-020)
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Source: SkinEthic Laboratories, Lyon, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 μl (undiluted) directly on top of the skin tissue

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hour post-incubation period

Number of replicates:

The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 replicates

Value:

32

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Since the mean relative tissue viability for GR-88-0778 was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.

Other effects / acceptance of results:

The positive control had a mean cell viability of 24% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

GR-88-0778 was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that GR-88-0778 did interact with the MTT endpoint. In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by GR-88-0778 was 3% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues. The mean absorption at 570 nm measured after treatment with GR-88-0778 and controls are recorded. The individual OD570 measurements are also recorded and shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-88-0778 compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-88-0778 compared to the negative control tissues was 32%. Since the mean relative tissue viability for GR-88-0778 was below 50% it is considered to be irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 24%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Finally, it is concluded that this test is valid and that GR-88-0778 is irritant in the in vitro skin irritation test under the experimental conditions described in the report.

Executive summary:

In vitro skin irritation test with GR-88-0778 using a human skin model.

This report describes the ability of GR-88-0778 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of GR-88-0778 was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch 6 of GR-88-0778 was a colourless to pale yellow liquid with a purity of 98.6%. GR-88-0778 was applied undiluted (25 μl) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

GR-88-0778 did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by GR-88-0778 was 3% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-88-0778 compared to the negative control tissues was 32%. Since the mean relative tissue viability for GR-88-0778 was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant. The positive control had a mean cell viability of 24% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that GR-88-0778 is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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:

Sep to Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: NYMPHEAL
Appearance: Colourless to pale yellow liquid
Batch: SC00018060
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 28 April 2017 (expiry date)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Albino rabbit, New Zealand White, (SPF-Quality)

Details on test animals or tissues and environmental conditions:

Number of animals 3 Males
Age and body weight At start of dosing, the animals were between 12 and 24 weeks old and body weights were at least 1.5 kg.
Identification Earmark.
Health inspection At least prior to dosing. It was ensured that the animals were healthy and that the eyes were free from any abnormality.
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation
Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.
Diet
Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period.
Water
Free access to tap water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.1 mL of Nympheal (100%)

Duration of treatment / exposure:

Single samples of 0.1 mL of NYMPHEAL were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation.

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 1 week later, after considering the degree of eye irritation observed in the first animal.
One hour prior to instillation of the test item, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test item, two drops of the topical anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.
Each animal was treated by instillation of 0.1 mL of the test item, in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) 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 to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection.
After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Irritation parameter:

cornea opacity score

Basis:

animal: 195

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal: 195

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal: 195

Time point:

24/48/72 h

Score:

1.3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 195

Time point:

24/48/72 h

Score:

0.7

Reversibility:

fully reversible within: 72hrs

Irritation parameter:

cornea opacity score

Basis:

animal: 211

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal: 211

Time point:

24/48/72 h

Score:

0.3

Reversibility:

fully reversible within: 48hrs

Irritation parameter:

conjunctivae score

Basis:

animal: 211

Time point:

24/48/72 h

Score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 211

Time point:

24/48/72 h

Score:

2.3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal: 212

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal: 212

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal: 212

Time point:

24/48/72 h

Score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 212

Time point:

24/48/72 h

Score:

1.3

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Irritation
Instillation of 0.1 mL of NYMPHEAL into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae. The effects on the cornea consisted of slight dulling of the normal luster of the cornea and epithelial damage with an area of 25% in one animal only. The corneal injury resolved within 72 hours. Iridial irritation was observed 24 hours after installation for one animal only. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in all animals.
Corrosion
There was no evidence of ocular corrosion.

Other effects:

Coloration / Remnants
No staining of (peri) ocular tissues by the test item was observed and no test item remnants were seen.
Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

List of Study Plan Deviations

1. Deviations from the maximum level of daily mean relative humidity occurred.

Evaluation: Laboratory historical data do not indicate an effect of the deviations.

The study integrity was not adversely affected by the deviation.

List of Standard Operating Procedures Deviations

Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on these results NYMPHEAL should be classified as Irritating to eyes (Category 2) according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

SUMMARY

Acute eye irritation/corrosion study with NYMPHEAL in the rabbit.The study was carried out based on the guidelines described in: OECD No.405 (2012) "Acute Eye Irritation / Corrosion" EC, No 440/2008, B5: "Acute Toxicity: Eye Irritation/Corrosion" EPA, OPPTS 870.2400 (1998): "Acute Eye Irritation" JMAFF Guidelines (2000), including the most recent revisions. Single samples of 0.1 mL of NYMPHEAL were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation. Instillation of NYMPHEAL into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae. The effects on the cornea consisted of slight dulling of the normal luster of the cornea and epithelial damage with an area of 25% in one animal only. The corneal injury resolved within 72 hours. Iridial irritation was observed 24 hours after installation for one animal only. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in all animals.

Based on these results NYMPHEAL should be classified as Irritating to eyes (Category 2) according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available and key results described in this summary, the substance has shown some skin and eye irritancy potential and should therefore be classified as Skin Irritant Category 2 and Eye irritant Category 2 according to the (EC) No 1272/2008 Regulation (CLP).