Reaction mass of glycerol, glycerol 1-formate, glycerol 2-formate, glycerol 1,2-diformate, glycerol 1,3-diformate and glycerol triformate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This endpoint study record is part of a Weight of Evidence approach comprising an in vitro BCOP assessment (this study) with the target substance and a read-across analogue approach which considered in vivo source substance information and are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

This endpoint study record is part of a Weight of Evidence approach comprising an in vitro BCOP assessment with the target substance and a read-across analogue approach (this record) which considered in vivo source substance information and are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Data for propane-1,2,3-triol (glycerol; CAS No. 56-81-5), formic acid, sodium salt (1:1) (sodium formate; CAS No. 141-53-7), formic acid, potassium salt (1:1) (potassium formate; CAS No. 590-29-4), formic acid, potassium salt (2:1) (potassium diformate; CAS No. 20642-05-1), formic acid, calcium salt (2:1) (calcium formate; CAS No. 544-17-2), 1,2-ethanediol, 1,2-diformate (ethylene diformate; CAS No. 629-15-2), 1,2-ethanediol (ethylene glycol; CAS No. 107-21-1) and formic acid (CAS No. 64-18-6) is used to address the toxicological data requirements for propane-1,2,3-triol and its esterification products with formic acid (EC No. 701-316-8) in an analogue read-across approach. The basis for this read-across approach is that, upon oral administration, the target substance is expected to undergo stepwise transformation by esterases in which the triformate constituent is converted into diformate constituents, diformate constituents are converted to monoformate constituents, and monoformate constituents to free glycerol, with formic acid being released at each step, as illustrated in Figure 1. The toxicity of the glycerol constituent/metabolite will be assessed using information on glycerol, and the toxicity of the formic acid metabolite will be assessed using information on sodium formate, potassium formate, potassium diformate, calcium formate, ethylene diformate, ethylene glycol and formic acid.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target Substance: Propane-1,2,3-triol and its esterification products with formic acid; 701-316-8
Source Substance 1: Propane-1,2,3-triol ; 200-289-5 ; 56-81-5
Source Substance 2: Formic acid, sodium salt (1:1) ; 205-48-0 ; 141-53-7
Source Substance 3: Formic acid, potassium salt (1:1) ; 209-677-9 ; 590-29-4
Source Substance 4: Formic acid, potassium salt (2:1) ; 243-934-6 ; 20642-05-1
Source Substance 5: Formic acid, calcium salt (2:1) ; 208-863-7 ; 544-17-2
Source Substance 6: 1,2-Ethanediol, 1,2-diformate ; 211-077-7 ; 629-15-2
Source Substance 7: 1,2-Ethanediol ; 203-473-3 ; 107-21-1
Source Substance 8: Formic acid ; 200-579-1 ; 64-18-6
[See attached JUSTIFICATION FOR READ-ACROSS OF TOXICOLOGICAL INFORMATION for further details.]

The TS is a UVCB substance of 100% purity. On this basis, the source substances collectively represent 100% w/w of the target substance. The purities of the samples of source substances that were tested are not specifically known, but it is assumed that they would not have been sufficiently impure as to substantially affect the study results. On this basis, the applicability of the data on the source substances to the TS is not expected to be compromised by the presence of impurities in any of the substances.

3. ANALOGUE APPROACH JUSTIFICATION
The basis for this read-across approach is that, upon oral administration, the TS is expected to undergo stepwise hydrolysis by esterases in which the triformate constituent is converted into diformate constituents, diformate constituents are converted to monoformate constituents, and monoformate constituents to the free glycerol constituent, with formic acid being released at each step.
The TS is manufactured from a 1:1 molar ratio of glycerol and formic acid reactants and will therefore metabolize back to those same proportions of those same reactants. An exposure of an organism to the TS is therefore considered to be essentially equivalent to an exposure to a 1:1 molar mixture of glycerol and formic acid.

Refer to the attached JUSTIFICATION FOR READ-ACROSS OF TOXICOLOGICAL INFORMATION for further details as to how the source substances relate to the target substance.

For the following toxicity endpoints:
8.2 serious eye damage / eye irritation
8.3 skin sensitisation
8.5.1 acute toxicity by oral route
8.4.1 in vitro gene mutation study in bacteria;
8.4.2 mutagenicity: in vitro cytogenicity study in mammalian cells;
8.4 mutagenicity: in vivo genotoxicity;
8.6.3 long-term repeated dose toxicity: ≥ 12 months; and
8.7.2 developmental toxicity
information on the glycerol constituent/metabolite and the formic acid metabolite of the target substance will be used to predict the properties of the target substance.

4. DATA MATRIX
Refer to the attached JUSTIFICATION FOR READ-ACROSS OF TOXICOLOGICAL INFORMATION for data matrix details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

other:

Irritation parameter:

other: Injury grade

Basis:

mean

Time point:

other: 18-24 h

Score:

5

Max. score:

10

Reversibility:

not specified

Remarks on result:

other: 0.005 mL undiluted gives injury up to 5.0 points (0.02 mL gives over 5.0)

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not determinable because of methodological limitations

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not determinable because of methodological limitations

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not determinable because of methodological limitations

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not determinable because of methodological limitations

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

On the bases that the BCOP assessment of the TS indicates the product is not corrosive yet possesses irritating potential and SS6 exhibited significant eye irritancy, a weight-of-evidence assessment allows for a conclusive decision on the classification of the TS such that the TS causes serious eye irritation.

Executive summary:

Results from an in vitro Bovine Corneal Opacity and Permeability (BCOP) study according to OECD Test Guideline 437 with the TS indicated no prediction could be made according to the UN GHS classification system. Additionally, the TS would be considered a mild irritant according to OECD Guidance Document No. 160. As the BCOP test method is not recommended for the identification of substances or mixtures that should be classified as irritating to eyes (CLP Category 2) further testing with another suitable method is required to conclusively determine the classification of the TS. As no other eye irritation data were available for the TS, data on the analogue source substances SS1, SS6 and SS7 were identified. The data on the analogue source substances and the in vitro BCOP results of the TS will be assessed to form a weight-of-evidence conclusive classification of the TS in regard to eye irritation potential.

The eye irritation potential of SS1, SS6, and SS7 was investigated in rabbits [5]. Albino rabbit eyes were selected on the basis of an absence of grossly visible staining by a 5% aqueous solution of fluorescein sodium, flushed with distilled water 20 seconds after application. After a 2-hour resting period, 0.005 mL of undiluted test substance was applied to the eyes. The eyes were examined in strong diffuse daylight, then stained with fluorescein, and the injury scored 18 to 24 hours following the application against a 1 (least) to 10 (worst) scale. Based on the results of the grading, additional applications of 0.02, 0.1 or 0.5 mL were made to assign the test substance to one of the grades. An application of 0.005 mL of undiluted SS6 resulted in an injury score of up to 5.0, and 0.02 mL of undiluted SS6 resulted in a score above 5.0 (where 5 represents severe injury). In contrast, applications of the maximum 0.5 mL dose of SS1 or SS7 only resulted in injury scores in the range of 0 to 1.0.

Since SS6, a formate ester, exhibited significant eye irritancy, whereas SS1 and SS7, both un-esterified alcohols, exhibited little or no eye irritancy, it is reasonable to conclude that the presence of the formate (whether in esterified form or as free formic acid/formate ion hydrolysis product) was responsible for the effect. Given that SS6 contains 203% of the formate in the same dose of TS, SS6 is taken to represent a worse case for eye irritancy than the TS.

On the bases that the BCOP assessment of the TS indicates the product is not corrosive yet possesses irritating potential and SS6 exhibited significant eye irritancy, a weight-of-evidence assessment allows for a conclusive decision on the classification of the TS such that the TS causes serious eye irritation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Reaction mass of glycerol, glycerol 1-formate, glycerol 2-formate, glycerol 1,2-diformate, glycerol 1,3-diformate and glycerol triformate

EC Number:

701-316-8

Molecular formula:

C5H8O5 & C4H8O4

IUPAC Name:

Reaction mass of glycerol, glycerol 1-formate, glycerol 2-formate, glycerol 1,2-diformate, glycerol 1,3-diformate and glycerol triformate

Specific details on test material used for the study:

Reported as: Glycerol Formates
Alternate name: Acidgen FG3

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

- Source: ABP, Perth, PH1 3XB, UK
- Storage, temperature and transport conditions of ocular tissue: Cow eyes were collected from freshly slaughtered cattle, and placed into containers containing Hank’s balanced salt solution (HBSS). Transport was on the same day as slaughter, and eyes were kept cool using cool packs during transport.
- Time interval prior to initiating testing: Corneas were prepared and used for testing on the day of collection.
- indication of any existing defects or lesions in ocular tissue samples: Eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL per cornea

Duration of treatment / exposure:

Dosed units were incubator for 10 minutes

Duration of post- treatment incubation (in vitro):

Recovery period of 2 hours

Number of animals or in vitro replicates:

Test item: 3 corneas
Negative control: 4 corneas
Positive control: 4 corneas

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use. Corneas from undamaged eyes were dissected, leaving a sclera margin of about 3 mm. Collected corneas were placed epithelial side down in HBSS at ambient temperature until use.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the equilibration period, the medium in both chambers was replaced with fresh, prewarmed Minimum Essential Medium (MEM) without phenol red. Baseline opacity readings were then taken. Damaged corneas (opacity >7 opacity units) were rejected from further use.

NUMBER OF REPLICATES
Test item: 3 corneas
Negative control: 4 corneas
Positive control: 4 corneas

NEGATIVE CONTROL USED
Negative control, physiological saline (750 μL)

POSITIVE CONTROL USED
Positive control, ethanol (750 μL)

APPLICATION DOSE AND EXPOSURE TIME
Application dose: 750 µL
Exposure time: 10 minutes

TREATMENT METHOD:
Corneas were mounted, epithelial side forward, into pre-warmed corneal holders. These holders consist of anterior and posterior compartments, which allow access to the epithelial and endothelial sides of the cornea, respectively. Once mounted, both chambers of the cornea holders were filled with pre-warmed MEM without phenol red. The posterior chamber was filled first to encourage the cornea to return to its original curvature, and any bubbles introduced into the medium were removed.

POST-INCUBATION PERIOD:
Holders were allowed to equilibrate for >1 h in an incubator.

REMOVAL OF TEST SUBSTANCE
MEM with and without phenol red were pre-warmed prior to use. Following the exposure incubation of 10 minutes, the test items and control solutions were removed from the anterior chambers. Corneas were then rinsed three times with MEM with phenol red (about 5 mL per rinse), removing the MEM each time, then rinsed once with MEM without phenol red (about 5 mL), which was also removed. Finally, both chambers were refilled with MEM without phenol red, and any bubbles introduced into the medium were removed.

POST-EXPOSURE INCUBATION:
A recovery period of 2 hours.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Same method as for the baseline opacity measurement
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)
- Others: Gross damage or other changes were observed by visual assessment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS ≤ 3 – UN GHS: No Category - OECD Guidance Document No. 160: Not classified
IVIS > 3 - ≤ 55 – UN GHS: No prediction can be made - OECD Guidance Document No. 160: > 3.1 - ≤ 25: Mild Irritant or > 25 - ≤ 55: Moderate Irritant
IVIS > 55 – UN GHS: Category 1 - OECD Guidance Document No. 160: Severe Irritant

DECISION CRITERIA: The decision criteria as indicated in OECD TG 437 were used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

5.75

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
Opacity Change (mean): 0.05
Permeability (A490) (mean): 0.004
Historical Data:
Opacity Change (mean): 4.24; Standard Deviation 2.01; Maximum: 8.54
Permeability (A490) (mean): 0.022; Standard Deviation 0.036; Maximum: 0.196

- Acceptance criteria met for positive control: Yes, IVIS (mean): 69.18
Historical Data: IVIS (mean) 69.36, acceptable Range 42.67 to 96.05

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The BCOP assay showed that no prediction could be made for Glycerol Formates according to the UN GHS classification system. Additionally, according to the classifications in OECD Guidance Document No. 160, Glycerol Formates was a mild irritant. On the bases that the BCOP assessment of the TS indicates the product is not corrosive yet possesses irritating potential and SS6 exhibited significant eye irritancy, a weight-of-evidence assessment allows for a conclusive decision on the classification of the TS such that the TS causes serious eye irritation

Executive summary:

The objective of this study was to evaluate the ocular irritation potential of Glycerol Formates using the Bovine Corneal Opacity and Permeability (BCOP) in vitro assay. Corneas were dissected from freshly obtained cow eyes, and mounted into corneal holders. Following a pre-dose equilibration period, and measurement of baseline opacity (using an opacitometer), the epithelial sides of the corneas were treated as follows:

 

• Glycerol Formates (750 μL) was applied to 3 corneas

• Physiological saline or ethanol (750 μL) as negative and positive controls, respectively, were applied to 4 corneas each

 

Following exposure for 10 min ± 1 min, the test or control items were rinsed off and corneas underwent a recovery period of 2 h ± 5 min.

 

The opacity of each cornea was then determined, followed by measurement of permeability by assessment of the passage of sodium fluorescein through the cornea. Irritancy was assigned on the basis of in vitro irritancy scores (IVIS). The Glycerol Formates mean IVIS Score is 5.75.

 

The BCOP assay showed that no prediction could be made for Glycerol Formates according to the UN GHS classification system. Additionally, according to the classifications in OECD Guidance Document No. 160, Glycerol Formates was a mild irritant.

 

Results from an in vitro Bovine Corneal Opacity and Permeability (BCOP) study according to OECD Test Guideline 437 with the TS indicated no prediction could be made according to the UN GHS classification system. Additionally, the TS would be considered a mild irritant according to OECD Guidance Document No. 160. As the BCOP test method is not recommended for the identification of substances or mixtures that should be classified as irritating to eyes (CLP Category 2) further testing with another suitable method is required to conclusively determine the classification of the TS. As no other eye irritation data were available for the TS, data on the analogue source substances SS1, SS6 and SS7 were identified. On the bases that the BCOP assessment of the TS indicates the product is not corrosive yet possesses irritating potential and SS6 exhibited significant eye irritancy, a weight-of-evidence assessment allows for a conclusive decision on the classification of the TS such that the TS causes serious eye irritation.