Graphene

Endpoint summary

Description of key information

Key: In vivo, skin sensitisation, guinea pig, OECD 406: not skin sensitising (Cada, 2020)

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:

30 Dec 2019 to 28 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted July, 1992

Deviations:

yes

Remarks:

There were occasional procedural deviations that were minor and did not impact the integrity or outcome of the study

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

1.No LLNA test (OECD TG 429) was conducted for the purpose of this registration according to Regulation (EC) No 1907/2006, as an OECD TG 406 study on Graphene nano platelets was already available as this study was explicitly requested for national registration under the Canadian Environmental Protection Act, 1999.

2.For the testing of Graphene nano platelets, the OECD TG 429 method is not applicable. The testing according to OECD 429 requires the test item to be suspended or dissolved in solvents/vehicles and subsequently applied on the mouse ear for incubation. Due to physical-chemical properties of Graphene, this procedure is not feasible. Graphene is an insoluble solid and it is difficult to keep the substance on the mouse ear for a sufficiently long time (it is also impossible to keep an e-collar on mice as well as to bandage for topical administration).
These technical difficulties will most likely result in minimal or no topical administration and possible oral administration in the LLNA. In contrast, test item application via an occlusive patch enables testing of such substances in the Buehler method.
All in all, due to topical dose administration difficulties in the mouse the OECD 406, Buelher test is the method of choice.

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 395–490 g
- Housing: Animals were housed individually with an enrichment device in polycarbonate cages with appropriate bedding.
- Diet: ad libitum (Envigo Teklad Certified Guinea Pig Diet)
- Water: ad libitum ( Municipal tap water)
- Acclimation period: an acclimation period of 7 days was allowed between receipt of the animals and the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): a light dark cycle of 12 hours light/12 hours dark, except during Study protocol-designated procedures

Route:

epicutaneous, occlusive

Vehicle:

other: saline 0.9%

Concentration / amount:

0.25 + 0.01 g

Day(s)/duration:

dosing on day 1, day 8 and day 15 / for 6 hours ± 15 minutes

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: saline 0.9%

Concentration / amount:

0.25 + 0.01 g

Day(s)/duration:

dosing on day 29 / for 6 hours ± 15 minutes

Adequacy of challenge:

other: highest technically applicable concentration used

No. of animals per dose:

treatment group: 20
naive control group: 10
positive control group: 10

Details on study design:

RANGE FINDING TESTS:
The test substance is not miscible in any substance and was found to be non-irritating at 0.5 g in a concurrent OECD 404 rabbit study, the maximal test amount according to the test guideline. For this skin sensitisation study in guinea pigs, 0.25 g was found to be the maximal achievable test amount to ensure adequate if not excessive dermal exposure during induction and challenge tests in this study in the same test area.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours ± 15 minutes
- Test groups: for the Graphene group, Graphene was applied (0.25 + 0.01 g) of gauze pre-wetted with 0.9% saline
- Control group: for the Naïve control group, 0.9% saline was applied; for the Positive control group, α-Hexylcinnamaldehyde was applied (0.4 mL)
- Site: dorsal flank area (Dose site 1)
- Frequency of applications: weekly (Days 1, 8, 15)
- Duration: 3 weeks
- Concentrations: 0.25 + 0.01 g Graphene

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 29
- Exposure period: 6 hours ± 15 minutes
- Test groups: for the Graphene group, Graphene was applied (0.25 + 0.01 g) of gauze pre-wetted with 0.9% saline
- Control group: for the Naïve control group, Graphene was applied (0.25 + 0.01 g) of gauze pre-wetted with 0.9% saline; for the Positive control group, α-Hexylcinnamaldehyde was applied (0.4 mL)
- Site: dorsal flank area (Dose site 2)
- Concentrations: same as induction phase
- Evaluation (hr after challenge): 24 and 48 h after removal of the dosing patch

Challenge controls:

naive control group: 10 male animals, Graphene was applied (0.25 + 0.01 g) of gauze pre-wetted with 0.9% saline on day 29
positive control group: 10 male animals, α-Hexylcinnamaldehyde was applied (0.4 mL) on day 29

Positive control substance(s):

yes

Positive control results:

During the induction phase, the majority of α-Hexylcinnamaldehyde exposed (positive control) animals demonstrated slight or defined erythema after dose administration.
The test conducted was found to be reliable as >15% of animals in the positive control group demonstrated slight or defined erythema during the challenge phase at a previously un-dosed site (site 2, 5 out of 10 animals or 50% responded).

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.25g

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No test substance-related clinical signs (for further information please refer to 'Any other inoframtion on results')

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.25g

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No test substance-related clinical signs (for further information please refer to 'Any other inoframtion on results')

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.25g

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No test substance-related clinical signs (for further information please refer to 'Any other inoframtion on results')

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.25g

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No test substance-related clinical signs (for further information please refer to 'Any other inoframtion on results')

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.4 mL

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

No test substance-related clinical signs (for further information please refer to 'Any other inoframtion on results')

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.4 mL

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

No test substance-related clinical signs (for further information please refer to 'Any other inoframtion on results')

Remarks on result:

positive indication of skin sensitisation

Clinical Observations:

There were no test substance-related clinical observations. Observations during the induction and challenge periods occurred in all dose groups (flaky skin), were found in a minimal number of animals (hair thinning or scab) or were expected based on the dose administration/wrapping procedure (yellow staining of the skin in the abdomen and/or perianal region).

Body Weights:

There were no test substance-related effects on body weights. All animals gained a satisfactory amount of weight during the induction and challenge periods and there were no statistical differences between any group at any time.

Dermal Scoring:

There were no test substance-related dermal effects observed. At all recorded time points, erythema and edema were scored as 0 at the dose site in both the control and the graphene dose groups during both the induction and challenge phases. During the induction phase, the majority of α-Hexylcinnamaldehyde exposed (positive control) animals demonstrated slight or defined erythema after dose administration.

The test conducted was found to be reliable as >15% of animals in the positive control group demonstrated slight or defined erythema during the challenge phase at a previously un-dosed site (site 2, 5 out of 10 animals or 50% responded).

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

Repeated dermal dosing of 0.25 g of graphene to male guinea pigs did not induce skin irritation during or after induction phase, and no dermal response was observed after challenge. These results show that graphene is not a skin sensitizer in guinea pigs.

Executive summary:

Study design

This study was conducted in compliance with the FIFRA Good Laboratory Practice Standards of 40 CFR Part 160 and the generally applicable principles of the OECD, ENV/MC/CHEM (98) 17 as well as any applicable amendments. This study was conducted according to guidelines OPPTS 870.2600 and OECD 406.

The objective of this study was to assess the skin sensitization potential of Graphene in guinea pigs. This information was utilized for the assessment and evaluation of the toxic characteristics of the test substance.

Induction or challenge dosing was administered according to the following table:

Group	No. of Males	Induction Dose Regimen	Induction Dose Test Substance^1	Challenge Dose Regimen	Challenge Dose Test Substance
1	10	1 time/week for 3 weeks (Days 1, 8, 15)	0.9% Saline	Day 29	0.9% Saline + 0.25 g Graphene
2	20	1 time/week for 3 weeks (Days 1, 8, 15)	0.9% Saline + 0.25 g Graphene	Day 29	0.9% Saline + 0.25 g Graphene
3	10	1 time/week for 3 weeks (Days 1, 8, 15)	0.4 mL α-Hexylcinnamaldehyde	Day 29	0.4 mL α-Hexylcinnamaldehyde

The dermal sensitization test was comprised of an induction and challenge phase. For the induction phase, the test substance was applied to an approximate 6 cm² area of skin of test substance group animals (n =20) every seven days for a total of three inductions (the same test site on the flank was used each time). The test substance remained in contact with the skin via closed patch occlusion for ~6 hours. A naïve control group (n= 10) was treated in the same manner during the induction phase with the exception that the control animals did not have test substance applied underneath the occlusive wrap. A positive control group (n= 10) was dosed concurrently with and in the same manner as the test substance-treated group. The positive control was α-Hexylcinnamaldehyde, a known mild-to-moderate sensitizer.

The challenge phase was completed two weeks after the last induction dose. The test substance-treated group (n= 20) and a naïve control group (n= 10) were dosed, as during the induction phase, with the test substance. These groups were treated on the opposite side from the application site for the induction phase. The dose sites were evaluated for erythema and edema at approximately 24 and 48 h after removing the challenge patch. The positive control group was dosed with α-Hexylcinnamaldehyde using the same procedures.

Results Multiple dermal exposure to graphene had no effect on mortality, clinical observations, dermal scoring, or body weights. The positive control test was validated by the presence of erythema in 50% of animals at a previously un-dosed skin site after challenge with α-Hexylcinnamaldehyde.

Conclusion

Repeated dermal dosing of 0.25 g of graphene to male guinea pigs did not induce skin irritation during or after induction phase, and no dermal response was observed after challenge. These results show that graphene is not a skin sensitizer in guinea pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Study design

This study was conducted in compliance with the FIFRA Good Laboratory Practice Standards of 40 CFR Part 160 and the generally applicable principles of the OECD, ENV/MC/CHEM (98) 17 as well as any applicable amendments. This study was conducted according to guidelines OPPTS 870.2600 and OECD 406.

The objective of this study was to assess the skin sensitization potential of Graphene in guinea pigs. This information was utilized for the assessment and evaluation of the toxic characteristics of the test substance.

The dermal sensitization test was comprised of an induction and challenge phase. For the induction phase, the test substance was applied to an approximate 6 cm² area of skin of test substance group animals (n =20) every seven days for a total of three inductions (the same test site on the flank was used each time). The test substance remained in contact with the skin via closed patch occlusion for ~6 hours. A naïve control group (n= 10) was treated in the same manner during the induction phase with the exception that the control animals did not have test substance applied underneath the occlusive wrap. A positive control group (n= 10) was dosed concurrently with and in the same manner as the test substance-treated group. The positive control was α-Hexylcinnamaldehyde, a known mild-to-moderate sensitizer.

The challenge phase was completed two weeks after the last induction dose. The test substance-treated group (n= 20) and a naïve control group (n= 10) were dosed, as during the induction phase, with the test substance. These groups were treated on the opposite side from the application site for the induction phase. The dose sites were evaluated for erythema and edema at approximately 24 and 48 h after removing the challenge patch. The positive control group was dosed with α-Hexylcinnamaldehyde using the same procedures.

Results

Multiple dermal exposure to graphene had no effect on mortality, clinical observations, dermal scoring, or body weights. The positive control test was validated by the presence of erythema in 50% of animals at a previously un-dosed skin site after challenge with α-Hexylcinnamaldehyde.

Conclusion

Repeated dermal dosing of 0.25 g of graphene to male guinea pigs did not induce skin irritation during or after induction phase, and no dermal response was observed after challenge. These results show that graphene is not a skin sensitizer in guinea pigs.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the key study for in vivo skin sensitisation, the registered substance is not subject to classification as skin sensitiser in accordance with Regulation (EC) No 1272/2008.