Reaction mass of bisisopropyl peroxydicarbonate and bis-sec-butyl peroxydicarbonate and isopropyl-secbutylperoxydicarbonate

Endpoint summary

Description of key information

Due to technical issues in vitro skin sensitisation testing with the multi-constituent substance is not possible.

There are no studies available for IPP, based on read across to CAS#16066-38-9 the substance is classified as not sensitising. SBP testing is underway, aKeratinosens assay is negative. A DPRA and H-CLAT are in progress due to the structural similarities with IPP the outcome of this study is assumed to be negative. The stabilizer has no sensitizing properties (section 13 QSAR doc appendix 1). QSAR data indicated the possibility of the multi-constituent substance to be a sensitizer (section 13 QSAR doc Appendix 2) based on data from another peroxide class; the acyl peroxides.

Based on the available information from studies, the multi-constituent substance is concluded not to be a sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 15-19, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Non animal test method -OECD approved. Activation of keratinocytes by the Induction of Antioxidant-
Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT) in the Keratinocyte ARE
Reporter Cell Line provides an in vitro procedure used for supporting the discrimination between skin
sensitizers and non sensitizers in accordance with the UN GHS.
According to REACH, In vivo methods can only be used if the in chemico or in vitro test methods are
not adequate for the substance or cannot be used for classification and risk assessment

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
-Source and lot/batch No of test material: Sponsor Lot No. 17121B3105
-Expiration date of the lot/batch:27 November 2018
Purity test date:10 February 2018
Purity:99.3%
Appearance:Clear colorless non-viscous liquid
Storage condition of test material:-15to -25 degrees celcius

Details on the study design:

Test system
The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using
MTT)
in the Keratinocyte ARE-Reporter Cell Line KeratinoSens skin sensitization assay is a
high-throughput cell based in vitro test to screen for the skin sensitization potential of chemicals.
The KeratinoSens cells (Givaudan, Switzerland) were propagated as a reporter cell line. The
KeratinoSens cells are transfected HaCaT keratinocytes that include a 56-base-pair insertion
containing
the ARE sequence from the AKR1C2 gene, a SV40 promoter, the luciferase gene in the vector pGL4
from Promega, and a stable insertion allowing for cells to be selected in the presence of Geneticin
(G418).
The signalling pathway with the repressor protein Keap1 and the transcription factor Nrf2, which binds
to the antioxidant / electrophile response element (ARE / EpRE), was shown to be a valuable cellular
endpoint to detect skin sensitizers in vitro. The induction of luciferase directly indicates the activation
of ARE dependent genes.
Cytotoxicity of a test article was assessed using cell MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyl
tetrazolium bromide). Cytotoxicity was determined by measuring the relative conversion of MTT
in test article-treated cultures compared to the solvent control at 570nm absorbance.
Experimental Design
The experimental design of this study consisted of three definitive assays to determine the maximal
induction (Imax), the concentration for maximal gene induction (CImax), the EC1.5 value (concen
tration for a statistically significant induction of 50% above solvent controls),
and a mean IC50 (concentration leading to 50% viability as compared to solvent controls) for the test
articles. For each
definitive assay, the KeratinoSens cells were cultured in quadruplicate plates for 24 hours, treated
with the test articles for 48 hours, and assessed for luciferase induction (3 plates) and cytotoxicity
(1 plate). The procedures that were performed in this assay were a modification of the procedures
previously described by Natsch, et al. (2008) and were performed similar to those procedures perfo
rmed by the Institute for In Vitro Sciences, Inc. in the KeratinoSens ring-study .
The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using M
TT) in the Keratinocyte ARE- Reporter Cell Line KeratinoSens Assay was performed to determine the
skin sensitization potential of the test articles, supplied by AkzoNobel.
The laboratory phase of this study was conducted from 15 to 19 April 2018 at the Institute for In
Vitro Sciences, Inc. (IIVS).
Evaluation of Test Results
A test article was predicted to have sensitization potential if:1) The EC1.5
value fell below 1000 μM or 200 μg/ml in at least 2 of 3 repetitions; 2) At the lowest concentration
with a gene induction above 1.5, cellular viability was greater than 70%; and 3) There was apparent
overall dose response which was similar between repetitions.

Positive control results:

The positive control cinnamic aldehyde had an EC 1.5 of <4 μM and IC50 of >64μM.

Key result

Run / experiment:

other: Mean of 3 definitive runs

Parameter:

other: EC 1.5 Value (uM)

Value:

2 000

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Gene fold induction above the solvent control There was no induction above 1.5 and cytotoxicity was greater than 70%

Other effects / acceptance of results:

Criteria for Determination of a Valid Definitive Assay
The KeratinoSens assay was accepted when the positive control (cinnamic aldehyde) caused an
EC1.5 value that fell within two standard deviations of the historical mean. Additionally, the results
of the three definitive trials for each plate are assessed using similar criteria outlined in the validation
ring trial . Those acceptance criteria included: 1) variability in DMSO solvent control wells for each
definitive assay was <20%; and 2) the positive control produced a statistically significant induction
above 1.5 fold below 64 μM in each definitive assay.

The test article, Di-sec-butyl peroxydicarbonate was tested in three definitive assays. Each definitive

assay included a

set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). The test article, was

tested at 12 concentrations ranging from 0.977 to 2000 μM. The positive control, Cinnamic Aldehyde,

was tested at 5 concentrations ranging from 4 to 64 μM. A summary of the EC1.5 (concentration

for a statistically significant induction of 50% above solvent controls) and IC(concentration leading to

50% viability as compared to solvent controls) results of the definitive assays are presented in Table

1. Additional luciferase induction information (which was not used for the current prediction model) that

includes the Imax(the maximal fold induction) and the CImax(the concentration at which the maximal

fold induction occurs), is also presented . A summary graph representing the luciferase fold induction

and the cell viability for each tested concentration of the test article is included .

A test article was predicted to have sensitization potential if: 1) The EC1.5 value fell below 1000 in at

least 2 of 3 repetitions; 2) At the lowest concentration with a gene induction above 1.5, cellular viability

was greater than 70%; and 3) There was an apparent overall dose response which was similar between

the three definitive assays.

According to the current prediction model, the test article, Di-sec-butyl peroxydicarbonate was predicted

to be a non sensitizer.

Interpretation of results:

GHS criteria not met

Remarks:

DPRA and H-CLAT still in progress

Conclusions:

Based on the data from The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT) in the Keratinocyte ARE- Reporter Cell Line KeratinoSens assay, the test article Di-sec-butyl peroxydicarbonate was predicted to be a skin non-sensitizer.

Executive summary:

The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT)

in the Keratinocyte ARE- Reporter Cell Line KeratinoSens was used to assess the skin sensitization

potential of the test article, Di-sec-butyl peroxydicarbonate (Lot# 17121B3105). The skin sensitization

potential of the test article was evaluated using the protocol that is consistent with the OECD Test

Guideline 442D “In VitroSkin Sensitisation: ARE-Nrf2 Luciferase Test Method”[1]. Based upon the

results of this study, the test article,Di-sec-butly peroxydicarbonate (Lot# 17121B3105) was classified

as a skin non sensitizer.

[1] OECD Test Guideline 442D “In VitroSkin Sensitisation: ARE-Nrf2 Luciferase Test Method)”,

Adopted 4 February 2015.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Aug-Nov 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Buehler method

Deviations:

yes

Remarks:

The dose preparations were not analyzed to confirm test article concentration, stability or homogeneity

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Study performed before the implementation of the REACH regulation

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, lnc., Haslett, Michigan.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: no data
- Weight at study initiation: males 347-411 g, females 329-375 g
- Housing: individually in suspended stainless steel cages
- Diet: PMI Certified Guinea Pig Chow #5026 (Purina Mills, lnc.) was provided ad libitum
- Water: Municipal tap water treated by reverse osmosis was available ad libitum
- Acclimation period: a minimum of five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-72
- Humidity (%): 53-77
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

Induction 1, day 0

Adequacy of induction:

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

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

75%

Day(s)/duration:

Induction 2 and 3, days 6 and 13

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

0.5%

Day(s)/duration:

Day 27

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

0.75%

Day(s)/duration:

day 34

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test: 10 males & females
Control: 5 males & females

Details on study design:

RANGE FINDING TESTS:
The test article was utilized at 100% and at 0.05%, 0.1 %, 0.25%, 0.5%, 1 %, 5%, 10%, 15%, 25%, 50% and 75% w/v in minerai oil for the range-finding studies.

Challenge controls:

no

Positive control substance(s):

yes

Remarks:

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

Positive control results:

Using Hexylcinnamaldehyde as a positive control, Springborn Laboratories, lnc., Spencerville, Ohio, has completed a study during the past six months which provided historical control data for contact sensitization to this agent utilizing the test system described herein (Modified Buehler Design). Following induction at 15% and 10% w/v and challenge at levels of 2.5% and 5% (mild concentrations) w/v Hexylcinnamaldehyde, a contact sensitization response was observed, thereby demonstrating the susceptibility of the test system to this sensitizing agent.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.5%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.5%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.5%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.5%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

0.75%

No. with + reactions:

2

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

0.75%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

0.75%

No. with + reactions:

1

Total no. in group:

10

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

0.75%

No. with + reactions:

1

Total no. in group:

10

Topical Range-Finding Studies

The results of the range-finding studies indicated that a test article concentration of 100% produced moderate irritation and was considered appropriate for induction. A concentration of 0.5% w/v in minerai oil was the highest non-irritating concentration; therefore, this concentration was considered appropriate for challenge.

Sensitization Study

Following induction 1 with 100% Lupersol 221 dermal scores of 1 to 3 (some with very slight to slight edema, blanching, and/or eschar and one with necrosis) were noted on 19/20 test animals. Due to the severe response, the concentration was reduced to 75% for inductions 2 and 3. Dermal scores of 2 to 3 (all with very slight to slight edema and some with blanching, superficial lightening and/or eschar) were noted for all test animals at both induction 2 and 3. Following challenge with 100% mineral oil dermal scores of 0 to ± were noted for all control animals. Following challenge with 0.5% w/v Lupersol 221 in minerai oil, dermal scores of 0 to ± were noted in all test and control animals. Group mean dermal scores were noted to be similar in the test animals as compared with the control animals. Following rechallenge with 0.75% w/v Lupersol 221 in minerai oil, dermal scores of 1 were noted in 2/20 test animals and in 1/10 control animals at the 24 hour scoring interval. At the 48 hour scoring interval, dermal scores of 1 were noted in 1/10 control animals. Dermal reactions in the remaining test and control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar to the test animals as compared with the control animals.

Interpretation of results:

GHS criteria not met

Conclusions:

Lupersol 221 is not considered to be a contact sensitizer in guinea pigs. The results of the Hexylcinnamaldehyde historical control study demonstrated that the test design utilized would detect potential contact sensitizers.

Executive summary:

The dermal sensitization potential of Lupersol 221 was evaluated in Hartley-derived albino guinea pigs. Ten male and ten female guinea pigs were topically treated with 100% Lupersol 221 (Induction I) or 75% w/v Lupersol 221 in mineral oil (Induction II and III), once per week, for three consecutive weeks. Five male and five female control guinea pigs were topically treated with 100% mineral oil, once per week for three consecutive weeks. Following a two-week rest period, a challenge was performed whereby the twenty test and ten control guinea pigs were topically treated with 0.5% w/v Lupersol 221 in mineral oil. Challenge responses in the test animals were compared with those of the control animals. Following a seven-day rest period, a rechallenge was performed whereby the twenty test animals and ten control guinea pigs were topically treated with 0.75% w/v Lupersol 221 in mineral oil. Rechallenge responses in the test animals were compared with those of the control animals. Lupersol 221: Following induction 1 with 100% Lupersol 221 dermal scores of 1 to 3 (some with very slight to slight edema, blanching, and/or eschar and one with necrosis) were noted on 19/20 test animals. Due to the severe response, the concentration was reduced to 75% for inductions 2 and 3. Dermal scores of 2 to 3 (all with very slight to slight edema and some with blanching, superficial lightening and/or eschar) were noted for all test animals at both induction 2 and 3. Following challenge with 100% mineral oil dermal scores of 0 to ± were noted for all control animals. Following challenge with 0.5% w/v Lupersol 221 in mineral oil, dermal scores of 0 to ± were noted in all test and control animals. Group mean dermal scores were noted to be similar in the test animals as compared with the control animals. Following rechallenge with 0.75% w/v Lupersol 221 in mineral oil, dermal scores of 1 were noted in 2/20 test animals and in 1/10 control animals at the 24 hour scoring interval. At the 48 hour scoring interval, dermal scores of 1 were noted in 1/10 control animals. Dermal reactions in the remaining test and control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar to the test animals as compared with the control animals.a-Hexylcinnamaldehyde: Usinga-Hexylcinnamaldehyde as a positive control, a study has been completed during the past six months which provided historical control data for contact sensitization to this agent utilizing the test system described herein (Modified Buehler Design). Following induction at 15% and 10% w/v and challenge at levels of 2.5% and 5% (mild concentrations) w/va-Hexylcinnamaldehyde, a contact sensitization response was observed, thereby demonstrating the susceptibility of the test system to this sensitizing agent. Based on the results of this study, Lupersol 221 is not considered to be a contact sensitizer in guinea pigs. The results of thea-Hexylcinnamaldehyde historical control study demonstrated that the test design utilized would detect potential contact sensitizers.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Not sensitizing based on SBP and IPP read across.