2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol

Endpoint summary

Description of key information

Di-pentaerythritol was found to be not irritating to skin in an in vitro SkinEthic EpiSkin assay. Di-pentaerythritol was also not irritating to eyes in vivo. 

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:

28.09.2009 to 22.01.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Proprietary GLP study conducted according to current guidelines

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline. In Vitro Skin Irritation: Reconstructed Human Epidermis (RHe) Test Method. 20 March 2009 (Version 6) 2nd Circulation.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Di-Penta 93, batch no. 3810066489, a white crystalline powder, was received from Perstorp Holding AB, and was stored protected from light at ambient room temperature. The Sponsor supplied Test Item Data Sheet stated that the batch was produced on 15 December 2008 and “expects not to expire within at least five years after production. The shelf-life is two years from dispatch.”

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human keratinocytes come from mammary/abdominal samples obtained from healthy consenting donors during plastic surgery.

Vehicle:

water

Details on test system:

The EpiSkin® reconstituted human epidermis models were obtained from Episkin SNC, Lyon, France. They are produced by culturing adult human keratinocytes on a collagen base in conditions which permit their terminal differentiation and the reconstruction of an epidermis with a functional horny layer (stratum corneum). The human keratinocytes come from mammary/abdominal samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2, HEP B and HEP C tests are carried out on the donors as well as verification of the bacteriological and fungal sterility of the cells. After a stage of immersed culture of the keratinocytes on the collagen support (3 days), during emersion (10 days) the epidermis differentiates and a horny layer is formed. The production of EpiSkin® is in accordance with the quality reference norms ISO 9001, ensuring traceability and reproducibility of the epidermal tissue as well as that of the quality control tests carried out on each batch of epidermis. The reproducibility of each batch is checked by histological analysis taking into account the general organisation, the stratification of the epidermis, the nucleation of the basal layer and the size of the intercellular spaces, adhesion of the basal layer to the support and the quantity of granular cells and the thickness of the horny layer. Each criterion is scored out of 4. The maximum score is 28. The minimum score for the criterion of acceptability of the model is 19.5. The reproducibility of the response of each EpiSkin® batch is tested against a reference irritant: SDS. The IC50 of the surfactantmn mn, is measured by the MTT assay after 18 h of contact. The minimum score for acceptability of the model is 1.5 mg/mL.

After arrival at Charles River, the EpiSkin® kits were inspected for quality. The pH and temperature indicators were both acceptable. Each individual unit was then transferred to a single well of a 12 well microtiter plate containing maintenance medium (2 mL). The maintenance medium had been warmed to ca 37°C in a waterbath. Prior to dosing, all units were then incubated for ca 24 h at ca 37°C in a humidified atmosphere with 5% CO2.

Prior to conducting the irritation assay, it was first necessary to determine if the test item was itself capable of reducing methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite. Since the assay depends on the ability of viable skin cells to reduce MTT to formazan, any reduction by the test item would interfere with the integrity of the results. The test item (ca 10 mg) was added to MTT (2 mL, ca 0.3 mg/mL) in PBS in a glass universal vial (3 replicates) and incubated at ca 37°C in a humidified atmosphere with 5% CO2 for ca 3 h. Formazan production was assessed by visual inspection. Three replicates of the positive control (eugenol, 10 μL) and the negative control (sterile, ultra-pure water, 10 μL) were tested in parallel to demonstrate the efficacy of the MTT solution

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg ± 2mg was applied onto the exposed surface of the EpiSkin. The surface of the EpiSkin was moistened with sterile, ultra-pure water (5 µL) prior to dosing. The surface area of the EpiSkin was ca 0.38 cm² and therefore the mean application rate was ca 23.6 mg/cm².

Duration of treatment / exposure:

Exposure: 15 minutes
Observation: 42 hours

Number of replicates:

Three viable EpiSkin units

Irritation / corrosion parameter:

% tissue viability

Remarks:

Percentage Viability of EpiSkin

Run / experiment:

Time point: 42 hours.

Value:

90.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Remarks: SD 5.19%.

Other effects / acceptance of results:

The negative control results were within the acceptance criteria defined in the ECVAM validation SOP.
The positive control results were within the acceptance criteria defined in the ECVAM validation SOP.
Exposure to Di-Penta 93 resulted in an EpiSkin® viability of 90.29% ± 5.19% of the negative control value. Percentage viabilities are shown in Table 1.

MTT Direct Reduction Test:

The test was scored by visual assessment of the formation of the purple-coloured formazan. The positive control (eugenol) reduced the MTT solution to formazan almost immediately, generating a dark purple colour before incubation. The negative control (sterile, ultra-pure water) and Di-Penta 93 did not reduce MTT to formazan after ca. 3 h incubation.

Table 1: Viability of EpiSkin Cultures

Test item	Mean Viability (%) after 42 hours	SD (%)	CV (%)
Di-Penta 93	90.29	5.19	5.75
5% SDS Solution (positive control)	10.40	4.10	39.42
PBS Solution (negative control)	100.00	7.82	7.82

Interpretation of results:

GHS criteria not met

Conclusions:

Di-Penta 93 is non-irritant when tested within the EpiSkin® in vitro irritation assay.

Executive summary:

Di-Penta 93 was tested for dermal irritation in vitro, using the SkinEthic EpiSkin® in vitro irritation assay. The endpoint of the assay was the estimation of cell viability by assaying the reduction of methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite by mitochondrial reductase. Irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value. A preliminary test was conducted to assess the ability of Di-Penta 93 to directly reduce MTT to formazan. Di-Penta 93 did not interact with the MTT solution. The irritation potential was assessed by applying Di-Penta 93 (ca 10 mg) directly onto the surface of three viable EpiSkin® reconstructed human epidermis units for ca 15 min. Di-Penta 93 was then washed from the surface of the EpiSkin® and the units returned to the incubator for a recovery period of ca 42 h. After the recovery period, the skin units were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for ca 3 h. Biopsies of the EpiSkin® membranes were then removed and added to acidic isopropanol. The formazan production was assessed by measuring the optical density of the extract at 550 nm and the viability of each individual tissue was calculated as a percentage of the mean negative control viability. Exposure to Di-Penta 93 resulted in an EpiSkin® viability of 90.29% ± 5.19% of the negative control value. The positive and negative controls, conducted in parallel, were within the defined acceptance criteria and demonstrated the efficacy of the test system.

In conclusion, Di-Penta 93 is non-irritant (“no category” in accordance with GHS classification) when tested within the EpiSkin® in vitro irritation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05.06. 2015 to 22.10.2015

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:

October 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Dipentaerythritol (2,2,2’,2’-tetrakis(hydroxymethyl)-3,3’-oxydipropan-1-ol)
- Physical state: crystalline powder
- Analytical purity: 94.6%
- Lot/batch No.: 150300133
- Expiration date of the lot/batch: 03 Mar 2017
- Storage condition of test material: ambient/dark
- pH: 4.53

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan UK, Oxon, England
- Age at study initiation: 12-15 weeks
- Weight at study initiation: 3.2-3.3 kg
- Housing: individually in appropriately sized stainless steel cages with a ‘Noryl’ dual level interior and perforated floor. Beneath each cage was a suspended tray containing absorbent paper. Objects for chewing were placed in each cage, and the cages contained a shelter for hiding in.
- Diet (e.g. ad libitum): Harlan diet, ad libitum
- Water (e.g. ad libitum): water from the public supply, ad libitum
- Acclimation period: up to 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18°C (target 16-20°C
- Humidity (%): 49-69% (target 40-70%)
- Air changes (per hr): at least 10/h (100% fresh air)
- Photoperiod (hrs dark / hrs light): 12 hour cycle

IN-LIFE DATES: From: 13 July 2015 To: 23 July 2015

Vehicle:

unchanged (no vehicle)

Controls:

other: contralateral eye remained untreated to serve as a control

Amount / concentration applied:

The test substance was administered as supplied. The weight equivalent of 0.1 mL was determined at the Test Facility and this weight was 79 mg.

Duration of treatment / exposure:

Not applicable - each animal received a single dose of the test substance, instilled into the lower conjunctival sac of the right eye. The lids were gently held closed together for 1-2 seconds.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): washing was not performed

SCORING SYSTEM: The scoring system detailed in OECD 405 (2012) was used; observations were made at 1, 4, 24, 48 and 72 hours after instillation.

TOOL USED TO ASSESS SCORE: hand-held magnifier and pen torch where necessary.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

There was no irritation noted at the cornea, iris or conjunctivae, and no ocular discharge was noted in either animal at any observation timepoint.

Other effects:

No other effects reported.

There was no irritation noted at the cornea, iris or conjunctivae, and no ocular discharge was noted in either animal at any observation timepoint. The mean values for each lesion, corneal opacity, iris, conjunctival redness and conjunctival chemosis were 0 (zero).

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was not irritating to rabbit eyes and classification is not required.

Executive summary:

The eye irritation potential of Dipentaerythritol was evaluated in New Zealand White rabbits according to OECD guideline 405. Two female rabbits were treated with the weight equivalent of 0.1 mL; determined to be 79 mg, of Dipentaerythritol, instilled into the lower conjunctival sac of the right eye. The left eye remained untreated and hence it acted as a control. Both eyes were examined for evidence of irritation approximately 1, 4, 24, 48 and 72 h after instillation. There was no irritation noted at the cornea, iris or conjunctivae, and no ocular discharge was noted in either animal at any observation timepoint. In conclusion, the instillation of Dipentaerythritol did not cause any ocular irritation to the rabbit eye. Based on the results of this study, Dipentaerythritol does not require classification for eye irritation according to CLP criteria.

Reference 2

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

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The in vivo eye irritation study was conducted prior to the adoption of the OCDE Guideline 492 on in vitro eye irritation in 2017.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Di-Penta 93 was tested for dermal irritation in vitro, using the SkinEthic EpiSkin® in vitro irritation assay (Blackstock, 2010). The study was conducted to GLP, and according to the Draft OECD Reconstructed Human Epidermis Test Method, and the ECVAM Skin Irritation Validation Study. The endpoint of the assay was the estimation of cell viability by assaying the reduction of methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite by mitochondrial reductase. Irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value. A preliminary test was conducted to assess the ability of Di-Penta 93 to directly reduce MTT to formazan. Di-Penta 93 did not interact with the MTT solution. The irritation potential was assessed by applying Di-Penta 93 (ca 10 mg) directly onto the surface of three viable EpiSkin® reconstructed human epidermis units for ca 15 min. Di-Penta 93 was then washed from the surface of the EpiSkin® and the units returned to the incubator for a recovery period of ca 42 h. After the recovery period, the skin units were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for ca 3 h. Biopsies of the EpiSkin® membranes were then removed and added to acidic isopropanol. The formazan production was assessed by measuring the optical density of the extract at 550 nm and the viability of each individual tissue was calculated as a percentage of the mean negative control viability. Exposure to Di-Penta 93 resulted in an EpiSkin® viability of 90.29% ± 5.19% of the negative control value. The positive and negative controls, conducted in parallel, were within the defined acceptance criteria and demonstrated the efficacy of the test system.

Eye irritation

The eye irritation potential of Dipentaerythritol was evaluated in New Zealand White rabbits according to OECD guideline 405. Two female rabbits were treated with the weight equivalent of 0.1 mL; determined to be 79 mg, of Dipentaerythritol, instilled into the lower conjunctival sac of the right eye. The left eye remained untreated and hence it acted as a control. Both eyes were examined for evidence of irritation approximately 1, 4, 24, 48 and 72 h after instillation. There was no irritation noted at the cornea, iris or conjunctivae, and no ocular discharge was noted in either animal at any observation timepoint. In conclusion, the instillation of Dipentaerythritol did not cause any ocular irritation to the rabbit eye.

Justification for selection of skin irritation / corrosion endpoint:
Only study available for this endpoint

Justification for selection of eye irritation endpoint:
Only study available for this endpoint

Justification for classification or non-classification

There was no evidence that di-pentaerythritol is irritating to skin or eyes. Therefore Di-pentaerythritol does not require classification as a skin or eye irritant according to the CLP Regulation.