Reaction mass of hexadecyl dihydrogen phosphate and cetyl alcohol (mono- C16 PSE and C16-OH)

Endpoint summary

Description of key information

Based on the available weight of evidence information from the read across studies of the main constituents, the test substance, ‘mono- C16 PSE and C16-OH’ is considered to be non-sensitiser to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From May 25, 2005 to June 13, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Source: Jackson Laboratories
Acclimation: 5 days
Number of animals: 37 females (nulliparous and non-pregnant)
Body weight: 16 - 21g
Body weight variation was within +/- 20% of the sex mean.
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Diet: Fresh PMI (Diet #5001)
Water: free access to tap water.

Vehicle:

other: Ultrapure liquid petrolatum

Concentration:

0, 2.5, 5, 10 and 25% w/w

No. of animals per dose:

5

Details on study design:

The test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, four experimental groups of five female CBA/J mice were treated with test substance concentrations of 2.5, 5, 10 or 25% w/w for three consecutive days, by topical application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Liquid petrolatum). A positive control group with a-hexylcinnamaldehyde (HCA - 50%) was also included in the experiment. Five days after the last exposure, all animals were injected with 5-bromo-2'-deoxy-uridine (BrdU) and the draining (auricular) lymph nodes were then isolated and pooled for each animal. Cells were fixed using 70% ethanol and used for the measurement of the cell number and the BrdU determination (percentage of proliferating cells in "S" phase). Flow cytometry was conducted for analysis and stimulation index (SI) was recorded

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

SI

Positive control results:

The SI value calculated for the positive control was 9.6 and ear swelling was observed in this group.

Key result

Parameter:

SI

Value:

ca. 0.9

Variability:

+/- 0.7

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

ca. 0.9

Variability:

+/- 0.6

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

ca. 0.7

Variability:

+/- 0.3

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

ca. 0.3

Variability:

+/- 0.1

Test group / Remarks:

25%

Cellular proliferation data / Observations:

- SI values were similar among the control and test groups and were below 3. Three concentrations (i.e.2.5, 5 and 25%) induced ear swelling.
- No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Based on the results of the read across study, the test substance, is considered to be non-sensitiser to the skin.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the read across substance, 'mono- and di- C16 PSE, K+ and H3PO4' (98.5%) according to OECD Guideline 429 and US EPA OPPTS 870.2600 (LLNA), in compliance with GLP. The read across substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, four experimental groups of five female CBA/J mice were treated with read across substance concentrations of 2.5, 5, 10 or 25% w/w for three consecutive days, by topical application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Liquid petrolatum). A positive control group with a-hexylcinnamaldehyde (HCA - 50%) was also included in the experiment. Five days after the last exposure, all animals were injected with 5-bromo-2'-deoxy-uridine (BrdU) and the draining (auricular) lymph nodes were then isolated and pooled for each animal. Cells were fixed using 70% ethanol and used for the measurement of the cell number and the BrdU determination (percentage of proliferating cells in "S" phase). Flow cytometry was conducted for analysis and stimulation index (SI) was recorded. Mortality/viability, body weights, clinical signs, ear size and irritation (and other local effects) were recorded as well. SI values were similar among the control and test groups and were below 3. Three concentrations (i.e.2.5, 5 and 25%) induced ear swelling. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period (MBRL, 2005). Based on the results of the read across study, the test substance, 'mono- C16 PSE and C16 -OH' is considered to be non-sensitiser to the skin.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test animals are initially exposed to the test substance by intradermal injection and/or epidermal application (induction exposure). Following a rest period of 10 to 14 d (induction period), during which an immune response may develop, the animals are exposed to a challenge dose. The extent and degree of skin reaction to the challenge exposure in the test animals is compared with that demonstrated by control animals which undergo sham treatment during induction and receive the challenge exposure.

GLP compliance:

not specified

Type of study:

other: modified Draize test

Justification for non-LLNA method:

Non LLNA justification
The in vivo study data were obtained in studies performed before any in vitro sensitization tests tests had been validated and accepted for regulatory purposes. Additionally, literature data demonstrates that an LLNA method is unreliable for surfactant substance, and may provide false positive results [1]. Therefore, an LLNA method is not deemed reliable for assessing the skin sensitisation of the substance.

[1]: Evaluating the sensitization potential of surfactants: Integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach. Ball et al. Regulatory Toxicology and Pharmacology 60 (2011) 389–400

Species:

guinea pig

Strain:

Hartley

Sex:

not specified

Details on test animals and environmental conditions:

Test animals:
- Source: not reported
- Weight at study initiation: ca 350 g
- Number of animals: 10
- Controls: only at rechallenge

Route:

intradermal

Vehicle:

not specified

Concentration / amount:

0.25%

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

other: Topical

Vehicle:

not specified

Concentration / amount:

10%

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

intradermal

Vehicle:

not specified

Concentration / amount:

0.1%

Day(s)/duration:

14 d after induction each animal received an intradermal injection in one flank and a topical application on the other

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

other: Topical

Vehicle:

not specified

Concentration / amount:

10%

Day(s)/duration:

14 d after induction each animal received an intradermal injection in one flank and a topical application on the other

Adequacy of challenge:

highest non-irritant concentration

Details on study design:

Administration / Exposure
- Study type: Non-adjuvant
- Preparation of test substance for induction: not reported, 0.1 mL of the test solution was administered.
- Induction schedule: 4 intradermal injections at one time point over the 2 auxillary and 2 inguinal lymph nodes.
- Concentrations used for induction: Based on a primary irritation screen the concentration used was 2.5 times the injection challenge concentration (the concentration giving slight barely perceptible irritation with no oedema).
- Challenge schedule: 14 days after induction each animal received an intradermal injection in one flank and a topical application on the other.
- Concentrations used for challenge: 0.1% intradermally ad 10% topically
- Rechallenge: Where materials test negative at challenge a repeat set of induction applications was carried out followed by challenge at 14 days and rechallenge (with controls) 7 days later.
- Positive control: not reported
- Pilot study: A preliminary irritation study was undertaken to determine the injection challenge concentration (the concentration giving slight barely perceptible irritation with no oedema) and the application challenge concentration (the highest concentration producing no irritation).

Examinations
- Grading system: A colour matching lighting unit was used to examine the skin reactions. Each injection reaction was scored based on size, erythema and oedema and considered positive if the total score was greater than the total average of the control scores. Application reactions were scored on a scale of 0 to +++ and considered positive if individual reactions were => + and there was no erythema in the controls.

Positive control substance(s):

not specified

Positive control results:

Results of the pilot study:
- 0.25%, 0.1% and 10% solutions were chosen for the intradermal induction, intradermal challenge and topical challenge respectively.

Results of test:
- Sensitization reaction: No sensitisation reactions at challenge or rechallenge following a second induction procedure. The result was reported as non-sensitising, individual animal data were not presented.
- Clinical signs: None
- Rechallenge: No sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.1% intradermal

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No sensitisation reactions

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10% topical

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No sensitisation reactions

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

other: not reported

Key result

Reading:

1st reading

Group:

negative control

Remarks on result:

other: not reported

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be non-sensitiser to the skin.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance, hexadecan-1-ol (purity:>95%), according to modified draize test in guinea pigs. Based on a primary irritation screening, 0.25%, 0.1% and 10% test substance solutions (the concentration giving slight barely perceptible irritation with no oedema) were chosen for the intradermal induction, intradermal challenge and topical challenge respectively. For induction phase, 0.1 mL of 0.25% intradermal and 10% topical application were performed. For challenge phase, 14 d after induction, each animal received  0.1% intradermal and 10% topical application of the test substance. Where materials test negative at challenge a repeat set of induction applications was carried out followed by challenge at 14 d and rechallenge (with controls) 7 d later. No positive control were reported. A colour matching lighting unit was used to examine the skin reactions. Each injection reaction was scored based on size, erythema and oedema and considered positive if the total score was greater than the total average of the control scores. Application reactions were scored on a scale of 0 to 3 and considered positive if individual reactions were ≥1 and there was no erythema in the controls. No sensitisation reactions at challenge or rechallenge following a second induction procedure were observed. There were no clinical signs were noted in all the animals. The study author had reported the test substance as non-sensitiser, whereas, the individual animal data were not presented. Under the study conditions, the test substance was determined to be non-sensitiser to the skin (OECD SIDS, 2006).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In absence of skin sensitisation study with the test substance, the endpoint has been assessed based on studies for substances representative of the two main constituents, which can be categorised as phosphate esters (PSE) and alcohol. The results are presented below:

Constituent 1: PSE - read across study:

A study was conducted to determine the skin sensitisation potential of the read across substance, ‘mono- and di- C16 PSE, K+ and H3PO4’ (assumed purity: 98.5%) according to OECD Guideline 429 and US EPA OPPTS 870.2600 (LLNA), in compliance with GLP. The read across substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, four experimental groups of five female CBA/J mice were treated with read across substance concentrations of 2.5, 5, 10 or 25% w/w for three consecutive days, by topical application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Liquid petrolatum). A positive control group with a-hexylcinnamaldehyde (HCA - 50%) was also included in the experiment. Five days after the last exposure, all animals were injected with 5-bromo-2’-deoxy-uridine (BrdU) and the draining (auricular) lymph nodes were then isolated and pooled for each animal. Cells were fixed using 70% ethanol and used for the measurement of the cell number and the BrdU determination (percentage of proliferating cells in “S” phase). Flow cytometry was conducted for analysis and stimulation index (SI) was recorded. Mortality/viability, body weights, clinical signs, ear size and irritation (and other local effects) were recorded as well. SI values were similar among the control and test groups and were below 3. Three concentrations (i.e.2.5, 5 and 25%) induced ear swelling. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Under the study conditions, the read across substance was considered to be non-sensitising to skin (MBRL, 2005).

Constituent 2: Alcohol:

A study was conducted to determine the skin sensitisation potential of hexadecan-1-ol (purity: >95%), according to the modified draize test in guinea pigs. Based on a primary irritation screening, 0.25%, 0.1% and 10% read across substance solutions (the concentration giving slight barely perceptible irritation with no oedema) were chosen for the intradermal induction, intradermal challenge and topical challenge respectively. For induction phase, 0.1 mL of 0.25% intradermal and 10% topical application were performed. For challenge phase, 14 d after induction, each animal received 0.1% intradermal and 10% topical application of the read across substance. Where materials test negative at challenge a repeat set of induction applications was carried out followed by challenge at 14 d and rechallenge (with controls) 7 d later. No positive control were reported. A colour matching lighting unit was used to examine the skin reactions. Each injection reaction was scored based on size, erythema and oedema and considered positive if the total score was greater than the total average of the control scores. Application reactions were scored on a scale of 0 to 3 and considered positive if individual reactions were ≥1 and there was no erythema in the controls. No sensitisation reactions at challenge or rechallenge following a second induction procedure were observed. There were no clinical signs were noted in all the animals. The study author had reported the read across substance as non-sensitiser. The individual animal data were not presented. Under the study conditions, the read across substance was determined to be non-sensitiser to the skin (OECD SIDS, 2006).

Overall, based on the available weight of evidence from studies on the main constituents, the test substance, ‘mono- C16 PSE and C16-OH’ is considered to be non-sensitiser to skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available weight of evidence information from the read across studies of the main constituents, the test substance, ‘mono- C16 PSE and C16-OH’ does not warrant classification for skin sensitisation according to EU CLP criteria (Regulation 1272/2008/EC).