Hexadecyl dihydrogen phosphate

Endpoint summary

Description of key information

Skin irritation: Under the conditions of this in vitro EpiDerm skin test, the test article, 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE), 100% Active; with a mean viability of 78.8%, is not considered an irritant.

Eye irritation: Under the conditions of this in vitro EpiOcular test, with a mean tissue viability of 2.1%, the test article, 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE), 100% Active; is considered an irritant. However, the actual classification i.e. Eye irritant Cat 2 or eye damage Cat 1 cannot be distinguished precisely. It is highly likely though that the very low result compared with the positive control (methyl acetate = eye irritant Cat 2) i.e. 2.1% verses 47.3%, that from a precautionary approach, this substance should be classified as Eye Damage Cat 1

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:

March 6, 2019 to March 8, 2019

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)

Version / remarks:

The MatTek Corporation In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) (OECD 439) Protocol: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT)
Protocol Date: 11/7/17

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE), 100% Active; Lot Number: 64252F18

Test system:

human skin model

Remarks:

The reconstructed human epidermal model EpiDerm consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

EpiDerm, when used with the recommended cell metabolism assay, can quickly provide toxicological profiles. The procedure utilizes a water-soluble, yellow, tetrazolium salt (MTT {3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide}), which is reduced by succinate dehydrogenase in the mitochondria of viable cells to a purple, insoluble formazan derivative. Substances which damage this mitochondrial enzyme inhibit the reduction of the tetrazolium salt. The amount of MTT reduced by a culture is therefore proportional to the number of viable cells

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

After the appropriate tissue preparation, milligrams of the test article, at 100%, 30 microliters of the positive control article and 30 microliters of the negative control article were each added to each of three Millicells containing the EpiDerm samples

Duration of treatment / exposure:

After dosing the last tissue, the six (6) well plates containing the dosed EpiDerm samples were incubated at 37°C, five (5)% carbon dioxide and > 95% humidity for 35 + 1 minutes.
After the 35 + 1 minute exposure period, the six well plates were removed from the incubator and placed in the sterile hood until the 60 minute exposure period was reached for the first dosed tissue.

Duration of post-treatment incubation (if applicable):

The EpiDerm samples were then returned to the incubator for 24 + 2 hours

Number of replicates:

Six

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three runs (6 plates)

Value:

78.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

As per MatTek and OECD 439, the assay meets the negative control criteria if the mean OD-570 for the negative control tissues is > 0.8 and < 2.8. The value is 1.473 and meets the acceptance criteria.
As per MatTek, the assay meets the positive control criteria if the mean viability of the positive control tissues, expressed as the percent of the negative control tissues is < 20%. The value is 3.4 and meets the acceptance criteria.
As per MatTek and OECD 439, the assay meets the standard deviation criteria if the standard deviation calculated from individual percent tissue viabilities of the 3 identically treated replicates is < 18%. The values are 14.11, 5.90 and 0.21 and they meet the acceptance criteria.

According to the EU and the Globally Harmonized System of Classification and Labeling of Chemicals, an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced to below 50% of the mean viability of the negative controls.

In vitro result                                                 In vivo prediction

Mean tissue viability < 50%                            Irritant, (R38 or GHS category 2)

Mean tissue viability > 50%                                   Non-irritant

Test Article Mean tissue viability = 78.8%              Non-irritant

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this test, the test article, 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE), 100% Active; Lot Number: 64252F18, at 100%, with a mean viability of 78.8%, is not considered an irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 20, 2019 to March 21, 2Ol9

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

The MatTek Corporation In Vitro EpiOcula Eye Initation Test (OCL-200-EIT) (OECD 492)
Protocol: Epi0cularrM Eye Iritation Test (OCL 200-EIT) Protocol Date: 6/29/15

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE),
100% Active; Iot Number:64252F18 (100%)

Species:

human

Strain:

other: stratified human keratinocytes

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 micrograms

Duration of treatment / exposure:

The tissues were incubated at standard culture conditions for 6 hours +/- 15 minutes.

Duration of post- treatment incubation (in vitro):

At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted offthe tissue, and the insert was blotted on absorbent material, and transfsned to the appropriate weli of the preJabeled Gwell plate containing I ml of warm Assay Medium. The tissues were incubated for 18 +/- 0.25 hours at standard culture conditions (Post-treatment Incubation).

Number of animals or in vitro replicates:

Duplicates

Details on study design:

*The EpiocularrM human cell construct (MatTek Corporation) is used in the assay. The use of EpiOcular cultures offers features appropriate for a model of ocular irritation. First, the model is composed of stratified human keratinocytes in a three-dimensional structure. Next, test materials can be applied topically to the model so that water insoluble materials may be tested . . . The toxicity of the test article (and thus the ocular initation potential) is evaluated by the relative
viability of the treated tissues relative to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal Enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test article-treated cultures."l

Irritation parameter:

other: % viability

Run / experiment:

1

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % viability

Run / experiment:

2

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

As per MatTek the assay meets the negative control criteria if the mean OD-570 for the negative control tissues is > 0.8 and < 2.5. The value is 1.764 and it meets the acceptance criteria.
As per MatTek and OECD 492,the assay meets the positive control criteria if the mean viability of the positive control tissues expressed as the percent of the negative control tissues is < 50%. The value is 47.3 and it meets the acceptance criteria
As per MatTek and OECD 492, the assay meets the acceptance criteria if the difference of viability between the two relating tissues of a single chemical is < 20% in the same run. The diflerences for all articles are below 20% and all meet the acceptance criteria.

The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.

The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (<=) to 60%. When the final mean percent tissue viability is less than or equal (<=) to 60% further testing with other test methods will be required because the Epiocular EIT shows a certain number of false positive results and cannot resolve between UN GHS Categories I and 2.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this test, with a mean tissue viability of 2.1%, the test article, 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE), 100% Active;
is considered an irritant. However, the actual classification i.e. Eye irritant Cat 2 or eye damage Cat 1 cannot be distinguished precisely.
It is highly likely though that the very low result compared with the positive control (methyl acetate = eye irritant Cat 2) i.e. 2.1% verses 47.3%, that from a precautionary approach, this substance should be classified as Eye Damage Cat 1

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)