Octan-4-olide

Endpoint summary

Description of key information

Skin irritation (in vitro): 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:

16 January 2018 - 17 April 2018

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: Chongqing Zoteq Aroma Chemical Co. Ltd; 170833910
- Expiration date of the lot/batch: 13 December 2019
- Purity:99.14%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
The test was carried out with the reconstituted three-dimensional human skin model EpiDer (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo (Appendix 1).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
The tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
After post-incubation, the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT (1 mg/mL) medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 h with gentle shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 min on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Historical data were generated from 2015 to 2017.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
To check the non-specific MTT-reducing capability of the test item 30 µL of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37  1 °C in the incubator. If the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. For quantitative correction of results, two killed tissues were treated with 30 µL of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. All steps were performed exactly as described in the chapter below. NSMTT was then calculated relative to the negative control of living tissues (NK) according to the following formula:

NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100
If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was corrected according to the following formula:

TODTT = ODTM – (ODKT – ODKU)
If non-specific MTT reduction is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.
To check the colouring potential of the test item 30 µL of the test item were mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min. If the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues treated with 30 µL of the test item (TVT). All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which were incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCliving) was then calculated according to the following formula:

NSCliving [%] = [ODTVT/ODNK]*100
If NSCliving is ≤ 5% relative to the negative control of living epidermis, no correction of the results is necessary.
If NSCliving is > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formula:
TODTT = ODTM – ODTVT

If NSCliving is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.
For test items which are classified as non-irritant and which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 30 µL of the test item (TKT). All steps were then performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which was incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCkilled) was then calculated according to the following formula:

NSCkilled [%] = [ODTKT/ODNK]*100
The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.
If the coloured test material or MTT reducing substance is classified as “irritant” i.e. mean tissue viability is < 50%, the correction procedures are not necessary.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”) if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance 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

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS solution

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours (24 ± 2 h and 18 ± 2 h)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Octan-4-olide

Value:

2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

5% SDS (positive control)

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not applicable

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None noted
- Direct-MTT reduction & colour interference with MTT: The mixture of 30 µL test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple.
The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.
The mixture of 30 µL of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8
- Acceptance criteria met for positive control: The mean relative tissue viability (% negative control) of the positive control was ≤ 20%
- Acceptance criteria met for variability between replicate measurements: Standard deviation of viability of replicate tissues of all dose groups was ≤ 18%

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In an in vitro skin irritation model (Epiderm), the mean relative tissue viability was ≤ 50% (2.0%) after 60 min treatment and 42 h post-incubation; therefire octan-4-olide is irritating.

Executive summary:

In an in vitro skin irritation assay in a human epidermal model EPIDERM (180396), reconstructed human epidermis tissue was exposed to 30 µL of octan-4-olide (99.14%) for 60 minutes. 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 2-hour 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 directly MTT reducing. The average viability of tissues treated by the test substance octan-4-olide was 2% compared to the negative control i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 3 % of negative control average value. According to these results, the test substance is 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:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17th April 2000 - 28th April 2000

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: Haarmann & Reimer GmbH; 50529270
- Expiration date of the lot/batch: Arfived on 20.03.2000; stable for 1 year
- Purity: 99.2%

Species:

rabbit

Strain:

other: SPF albino

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: BI Pharma KG, 88397 Biberach
- Weight at study initiation: 2.0 - 2.7 kg b.w.
- Housing: Individually in PPO cages (floor area: 2576 sq.cm) with perforated floor
- Diet: A pelleted complete rabbit diet "Altromin 2123" from Altromin, D-32791 Lage, Lippe, wasavailable ad libitum
- Water: The animals had free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 10 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness

Vehicle:

unchanged (no vehicle)

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:

24 hrs

Number of animals or in vitro replicates:

4 female rabbits

Details on study design:

SCORING SYSTEM: Draize system

TOOL USED TO ASSESS SCORE:
The day before testing both eyes of the animals were examined with a hand held inspection lamp fitted with white and UV-light and magnifying glass with 2 x magnification to ensure there were no defects or irritation. The examination was performed before and after instillation of Fluorescein. Only the left eye was treated. The right eye remained untreated and served as control. 0.1 ml of the test article was placed in the left eye of the rabbits by gently pulling the lower lid away from the eyeball to form a cup into which the test article was dropped. The lids were then gently held together for 1 second. The eyes were examined and the grade of ocular reaction was recorded one hour and 24 hours later. After the first 24 hour reading Fluorescein was instilled. After rinsing with 20 ml 0.9% sodium chloride solution the eyes were examined again using UV-light to detect possible corneal darnage. The eyes were also examined 48 and 72 hours as well as 7 days after the treatment.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hrs

Remarks on result:

other: after instillation of Fluorescein

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hrs

Irritation parameter:

cornea opacity score

Basis:

animal: 2, 3, 4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: 2, 3, 4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: 2, 3, 4

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 hrs

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hrs

Irritation parameter:

chemosis score

Basis:

animal: 3, 4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0.1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Time point:

24/48/72 h

Score:

0.8

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.2

Max. score:

4

Reversibility:

fully reversible

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo eye irriation studies in rabbits, octan-4-olide was not irritating.

Executive summary:

In a primary eye irritation study (2000072), 0.1mL of octan-4-olide (99.2%) was instilled into the conjunctival sac of the left eye of 4 female SPF albino rabbits. The eyes were examined and graded according to Draize at 1h, 24h, 48h, 72h, and on day 7 (one rabbit) and monitored up to 14 days.  

Twenty-four hours after application of the test article animal 1 showed a scattered or diffuse area of opacity on more than half, but less than three quarters of cornea, a diffuse crimson red conjunctiva with individual vessels not easily discemible and a swelling above normal. In animal 2 were observed some conjunctival vessels definitely injected and a swelling above normal. Animals 3 and 4 showed some conjunctival vessels definitely injected. Forty-eight hours after application of the test article all four animals showed some conjunctival vessels definitely injected. Seventy-two hours after application of the test article animal 1 showed some conjunctival vessels definitely injected. The other three animals were free of any signs of eye irritation. Seven days after application of the test article anirnal 1 was free of any signs of eye irritation, too.

The following mean values were obtained: cornea opacity 0.1; iris lesion 0.0; redness of conjunctiva 0.8 and oedema of conjunctiva (chemosis) 0.2

In this study, octan-4-olide is not an eye irritant to the eye based on the the current UNSCEGHS Guidance (adopted in June 2011).

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 test available for octan-4-olide.

In an in vitro skin irritation assay in a human epidermal model EPIDERM (OECD 439/GLP), reconstructed human epidermis tissue was exposed to 30 µL of octan-4-olide (99.14%) for 60 minutes. 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 2-hour 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 directly MTT reducing. The average viability of tissues treated by the test substance octan-4-olide was 2% compared to the negative control i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 3 % of negative control average value. According to these results, the test substance is irritating.

Serious eye damage/eye irritation:

There is no in vitro eye irritation test available for octan-4-olide. There is an in vivo eye irritation study in rabbits available.

In a primary eye irritation study (OECD 405/GLP), 0.1mL of octan-4-olide (99.2%) was instilled into the conjunctival sac of the left eye of 4 female SPF albino rabbits. The eyes were examined and graded according to Draize at 1h, 24h, 48h, 72h, and on day 7 (one rabbit) and monitored up to 14 days.  Twenty-four hours after application of the test article animal 1 showed a scattered or diffuse area of opacity on more than half, but less than three quarters of cornea, a diffuse crimson red conjunctiva with individual vessels not easily discemible and a swelling above normal. In animal 2 were observed some conjunctival vessels definitely injected and a swelling above normal. Animals 3 and 4 showed some conjunctival vessels definitely injected. Forty-eight hours after application of the test article all four animals showed some conjunctival vessels definitely injected. Seventy-two hours after application of the test article animal 1 showed some conjunctival vessels definitely injected. The other three animals were free of any signs of eye irritation. Seven days after application of the test article anirnal 1 was free of any signs of eye irritation, too.

The following mean values were obtained: cornea opacity 0.1; iris lesion 0.0; redness of conjunctiva 0.8 and oedema of conjunctiva (chemosis) 0.2. Four animals were used in this study. According to the Guidance on the Application of the CLP Criteria (Version 5.0 – July 2017), the current UNSCEGHS Guidance needs to be applied (adopted in June 2011). Using this criteria, octan-4-olide is not an eye irritant.

The results from these studies are acceptable to use in the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance octan-4-olide (CAS No. 104-50-7) is classified as Skin irritation Category 2 for skin irritation/corrosion and does not need to be classified for serious eye damage/eye irritation when considering the criteria outlined in Annex I of 1272/2008/EC.