2-ethylhexyl nitrate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 5 February 1998 to 27 May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Study was completed in 1998 prior to widespread regulatory acceptance of LLNA

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeder Charles River (France)
- Age at study initiation: approximately 3 months old
- Weight at study initiation: mean body weight +/- standard deviation of 329 g +/- 17 g for males and 342 g +/- 11 g for females
- Housing: polycarbonate cages
- Diet (e.g. ad libitum): 106 pelleted diet
- Water (e.g. ad libitum): drinking water filtered by a F.G Muillipore membrane (0.22 microns)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C +/- 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From 10 February 1998 to 13 March 1998

Route:

other: epicutaneous

Vehicle:

unchanged (no vehicle)

Concentration / amount:

Test item administered undiluted.

Route:

other: epicutaneous

Vehicle:

unchanged (no vehicle)

Concentration / amount:

Test item administered undiluted.

No. of animals per dose:

10 males and 10 females for treated group, 5 males and 5 females for control group

Details on study design:

RANGE FINDING TESTS:
Yes, to determine the concentrations to be used:
- the maximal practicable concentration or concentration causing weak to moderate skin reactions for induction
- the highest concentration which does not cause irritant effect.
The test item was used undiluted during the inductions and challenge phases.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours for each application
- Test groups: treated with the test substance (0.5 mL)
- Control group: treated with purified water (0.5 mL)
- Site: anterior flank
- Frequency of applications: days 1, 8 and 15
- Concentrations: substance used undiluted

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 29
- Exposure period: 6 hours
- Test groups: treated with the test substance (0.5 mL) on the right flank and with purified water (0.5 mL) on the left flank
- Control group: treated with the test substance (0.5 mL) on the right flank and with purified water (0.5 mL) on the left flank
- Site: posterior right and left flank
- Concentrations: substance used undiluted
- Evaluation (hr after challenge):

OTHER:

Positive control substance(s):

yes

Remarks:

2,4-dinitro chlorobenzene (DNCB)

Positive control results:

33% of the animals (3/10) showed a positive reaction with the DNCB.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No clinical signs and no death were noted

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs and no death were noted.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No clinical signs and no death were noted

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No clinical signs and no death were noted.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.5% w/w

No. with + reactions:

6

Total no. in group:

9

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.5% w/w

No. with + reactions:

3

Total no. in group:

9

Clinical observations:

Dryness of skin in 3 animals

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under the experimental conditions of the study and according to the Buehler method, the test substance 2-ETHYLHEXYLE NITRATE does not induce delayed contact hypersensitivity in guinea-pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A total of 6 skin sensitization assays were available within the SIEF. After thorough review by RSS/CSR author, incl. if necessary change of the original conclusions, four tests were of appropriate reliability, two otherswere K3, one of which was not included in IUCLID because it was not owned by a SIEF member ; all 6 tests are presented below.

Summary of key reliability and sensitivity features of the 6 skin sensitisation assays available to the 2-ethylhexylnitrate SIEF:

Reference	Batch (purity)	Sensitivity of the study*	Comments on sensitivity and reliability check	Original conclusion	Revised conclusion	Revision based on (results for undiluted product)**
J. Clouzeau (1988)	op 3/85 (NR)	Maximal (MK; 0.1 mL, 50%, 100%, 100%)	GLP, induction phase insufficiently described, no positive control: K2- but gives useful histopathology data to understand nature of skin reactions	Negative	Negative	No histopathological signs of sensitisation, overrides the 12/20 persistent macroscopic reactions at challenge
S.R. Kynoch, B.I. Parcell (1989)- report 89652D	B (NR)	Maximal (MK; 0.1 mL, 30%, 100%, 100+50%)	non-GLP, induction phase insufficiently described, 2/10 controls are positive** at challenge: K3***	NR	NA	7/20 persistent macroscopic reactions at challenge, biased by invalid controls
S.R. Kynoch, B.I. Parcell (1989)- report 89651D	A (>99%)	Medium (MK; 0.1 mL, 5%, 100%, 100+50%)	non-GLP, induction phase insufficiently described but no impact as turns positive: K2	NR	Positive	9/20 persistent macroscopic reactions at challenge
V M Davison (1988)	CI-0801 (NR)	Medium (MK; 0.05-0.1 mL, 10%, 100%, 100+30%)	non-GLP, purity unknown; induction phase insufficiently described but no impact as turns positive: K2	Positive	Positive	15/18**** persistent macroscopic reactions at challenge
X. Manciaux (1998)	0020/98 (99.84%)	Low (Buehler 3; NA; NA; 100%; 100%)	GLP, adequate detail level and protocol: K1- but less sensitive method (Buehler 3 applications)	Negative	Negative	0/20 macroscopic reactions at challenge
S.M. Glaza (1988)	NR (>99%)	Medium (MK; 0.05mL, 5%, 100%, 50%)	GLP, induction not maximized, leaving doubt about negative responses: K3	Negative	NA	0/20 macroscopic reactions at challenge, biased by non-maximized protocol

NR: not reported;       NA: not assessable or applicable

underlined: limited exposure inappropriately justified and/or deviating from the guideline

*: indicated in parentheses: method (MK or Buehler 3), volume for intradermal injection, concentrations for intradermal injection, topical induction and challenge

**: local or extensive reaction, at least until 48h after end of challenge, was re-counted as positive by RSS/CSR author

***: study not present in IUCLID dossier

****: animals with bandage slipped were excluded

Out of the four Klimisch 1-2 tests, three MK assays clearly involved macroscopic persistent reactions in >30% of challenged animals and a fourth test, a Buehler test, showed no reaction in any animal. Three main hypotheses could explain this apparent inconsistency:

- Inconsistencies could be related to an impact of sensitizing impurities and/or inter-lab variability: it was impossible to localize, decades afterwards, data on impurities in the tested batches; however, it is notable that studies by Clouzeau and Manciaux were done in the same lab for the same Sponsor, limiting this source of variability; furthermore, the synthesis process is mostly similar for all producers and unlikely to introduce a sensitizing impurity.

- Reactions could be irritation due to a bad protocol (too high concentrations): this is not likely if the test is correctly carried out (choice of a non-irritant challenge dose-level), and has been taken into account in Klimisch rating and review of conclusions.

- Reactions could be non-related to sensitization: this hypothesis is supported by four different points:

1) Study by Clouzeau showed macroscopic skin reactions sufficient for classification as sensitizer, but as opposed to all 5 other tests, this one included an histopathology confirmation that evidenced lesions evocating irritation (acanthosis and hyperkeratosis) and an absence of inflammatory cells, so it was concluded to be negative.

2) The substance, being an organic nitrate, has vasodilation properties (as confirmed by observation of headaches and dizziness in some workers) which could account for reddening of the skin that does not reflect the mechanism of standard irritation or sensitisation, but can be mistaken for it by macroscopic examination.

3) Skin cracking has been noted in rabbits after repeated dermal exposure (see 7.3) and may be related to interaction with skin lipids.

4) The IUCLID4 file (see attached file) of the synthesis precursor 2EH, which may be present as an impurity in some studies (no data), is attached and shows that this substance is clearly a skin irritant, so a possible hypothesis could be delayed formation of a skin irritant compound (in the tested batch or during the application time) leading to delayed skin irritation, misinterpreted as being sensitization.

A LLNA assay was not carried out to clarify the issue because it was considered unethical in the existence of 6 sensitisation assays (incl. 4 reliable ones), and one test would be unable to answer on the possible impact of impurities.

It may also be noted that the OECD QSAR Toolbox predicts an absence of protein binding, also in favour of an absence of sensitizing properties. See the attached document.

Migrated from Short description of key information:

A total of 6 skin sensitization assays were available within the SIEF. After thorough review by RSS/CSR author, four tests were of appropriate reliability, out of which 2 were concluded to be negative and 2 to be positive. One of the negative tests would have been considered positive due to macroscopic skin reactions, but was considered not to represent sensitisation but irritation (possibly delayed) due to an absence of typical inflammatory response at histopathology. This could also account for the other "positive" results in tests not including histopathological confirmation.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Migrated from Short description of key information:

No data. The substance is not a skin sensitiser and does not include structural alerts for respiratory sensitisation. It is therefore unlikely to be a respiratory sensitizer.

Justification for classification or non-classification

As the test item induced delayed skin reactions which:

- are not representative of sensitisation (inflammatory cell infiltration) after histopathological examination,

- may be related to vasodilation (the substance being an organic nitrate), interaction with skin lipids, or delayed formation of a skin irritant impurity/degradation product,

And in the absence of predicted protein binding according to OECD QSAR Toolbox,

the substance is considered as non-sensitizing as a weight-of-evidence.