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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 September to 5 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP study conducted according to current guidelines
Qualifier:
according to
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. certificate)
Species:
human
Strain:
other: not applicable.
Details on test animals and environmental conditions:
This was an in vitro study using the EpiSkin system, produced from human keratinocytes obtained from healthy donors..
Type of coverage:
open
Preparation of test site:
other: not applicable, the study was in vitro
Vehicle:
water
Controls:
other: The negative control was phosphate-buffered saline, the positive control was 5% sodium dodecyl sulphate (SDS)
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:
15 minutes
Observation period:
42 hours
Number of animals:
Three viable EpiSkin units
Details on study design:
After exposure to the test item or positive and negative controls for ca 15 min, the EpiSkin units were washed by rinsing with Dulbecco’s phosphate buffered saline (25 mL). After washing the units were blotted dry with tissue paper and transferred to fresh maintenance medium. After washing, all skin units were blotted dry with tissue paper, transferred to fresh maintenance medium and incubated for ca 42 h at ca 37°C in a humidified atmosphere with 5% CO2.

After the recovery period, all EpiSkin units were transferred to a new 12 well microtiter plate containing MTT solution (2 mL per well, ca 0.3 mg/mL) in assay medium. Each unit was tapped gently on tissue paper to remove residual moisture before transferring to the MTT solution. The skin units were then incubated for ca 3 h at ca 37°C in a humidified atmosphere with 5% CO2. After incubation, each skin unit was removed from the MTT solution and gently tapped dry on tissue paper to remove any excess moisture. The exposed skin was then completely removed from the unit using a specially designed biopsy punch. The epidermis was separated from the collagen matrix and both layers added to an appropriately labelled microfuge tube containing acidic isopropanol (500 μL). Samples were then stored at ca 4°C protected from light.
After ca 72 h storage, the samples were removed from the refrigerator and mixed by vortexing to ensure a homogenous mixture. Two aliquots (200 μL) of each sample were then added to a 96 well flat bottom microtiter plate for each sample, ensuring that no solid material was removed from the sample tube. Plates were analysed using an MRX plate reader using wavelength measurement at 550 nm. Absorbance values were calculated against the background acidified isopropanol sample contained on the plate.
Irritation / corrosion parameter:
other:
Value:
90.29
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 hours. Max. score: 94.39. Reversibility: other: not applicable. Remarks: SD 5.19%. (migrated information)
Irritant / corrosive response data:
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.
Other effects:
No other effects reported.

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.

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:
not irritating
Remarks:
Migrated information GHS no category Criteria used for interpretation of results: EU
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
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 June to 24 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study with no deviations
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
October 2012
Deviations:
no
GLP compliance:
yes
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:
other: mean 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: mean 24, 48 and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: mean 24, 48 and 72 hours
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: mean 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: mean 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: mean 24, 48 and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: mean 24, 48 and 72 hours
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: mean 24, 48 and 72 hours
Score:
0
Max. score:
4
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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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.

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.