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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-29 to 2017-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
OO-tert-butyl monoperoxymaleate
EC Number:
217-691-1
EC Name:
OO-tert-butyl monoperoxymaleate
Cas Number:
1931-62-0
Molecular formula:
C8H12O5
IUPAC Name:
(2Z)-4-(tert-butylperoxy)-4-oxobut-2-enoic acid
Specific details on test material used for the study:
- Name: OO-tert-butyl monoperoxymaleate
- CAS No.: 1931-62-0
- Batch No.: M-151001
- Physical State: solid
- Colour: white
- Purity: 47.3% peroxide (range 45-50 %); rest phlegmatizer (Triacetin, CAS: 102-76-1)
- Storage Conditions: maximum + 30 °C

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: The animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Age at study initiation: 8-9 weeks
- Weight at study initiation: between 16.5 and 21.3 g
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): ad libitum (Altromin 1324 maintenance diet for rats and mice)
- Water (e.g. ad libitum): ad libitum (tap water, sulphur acidified to a pH value of approx. 2.8)
- Acclimation period: at least five days

ENVIRONMENTAL AND HOUSING CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Photoperiod (hrs dark / hrs light): 12/12
- Air change: at least 10 x/hour


Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 3.125%, 6.25% and 12.5% (w/v)
No. of animals per dose:
- Main experiment: 5 mice/ group
Details on study design:
See "Any other information on materials and methods incl. tables" below.
Positive control substance(s):
other: phenylendiamine

Results and discussion

Positive control results:
A shared positive control was performed concomitantly; using 5 animals. The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system (see Table 2 in box "Any other information on results").

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
9.5
Variability:
SD = 3.0
Test group / Remarks:
3.125%
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
10.5
Variability:
SD = 4.1
Test group / Remarks:
6.25%
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
15
Variability:
SD = 2.8
Test group / Remarks:
12.5%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Please see Table 3 in box "Any other information on results"

EC3 CALCULATION
- The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were above 3.

CLINICAL OBSERVATIONS:
- The animals treated with the test item at a concentration of 6.25% and 12.5% showed erythema grade 1 on day 3 which is not considered a sign of excessive irritation. There were no significant clinical observations in any of the animals treated with the test item at a concentration of 3.125 % or the negative control. Neither mortality nor any effects on body weights were observed in any of the animals.

BODY WEIGHTS
- All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Any other information on results incl. tables

Results of the pre-screen tests

Solubility tests

The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO.

Irritation and toxicity test

One of the animals treated with the undiluted test item showed signs of systemic toxicity such as slight reduction in the spontaneous activity, secretion from the eyes on day 4. The local effects at the application sites revealed sticky fur and produced distinctive odour. The animal was found dead on day 6 of the study. The second animal treated with the undiluted test item showed signs of excessive irritation (increased ear thickness > 25%) on day 6 at both application sites. One of the animals treated with the diluted test item at a concentration of 50% showed signs of excessive irritation (increased ear thickness > 25%) on day 6 at the left application site.

One of the animals treated with the diluted test item at a concentration of 25% showed signs of excessive irritation (increased ear thickness > 25%) on day 6 at the left application site. According, to what was observed, the pre-screen test was concluded. All other clinical findings were considered not to be signs of systemic toxicity or signs of excessive irritation.

All surviving animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the pre-screen test.

Results of the main study

Table 2: Stimulation Indices obtained for the Positive-Control Group

Test Item Concentration
[%]
Animal Number Stimulation Index
Negative Control
AOO
100 101
102
103
104
105
MV 1.0
SD
Positive Control
Phenylenediamine
1 106 7.8
107 4.7
108 3.5
109 5.7
110 8.7
MV 6.1
SD 1.9

SD = standard deviation; MV = mean value

Table 3: Stimulation Indices obtained in the main experiment

Test Item Concentration
[%]
Animal Number Stimulation Index
Negative Control AOO 100 101
102
103
104
105
MV 1.0
SD
OO-tert-butyl monoperoxy-maleate in AOO 3.125 1 5.8
2 12.5
3 13.5
4 6.8
5 9.1
MV 9.5
SD 3.0
OO-tert-butyl monoperoxy-maleate in AOO 6.25 6 3.5
7 10.8
8 12.6
9 15.8
10 9.9
MV 10.5
SD 4.1
OO-tert-butyl monoperoxy-maleate in AOO 12.5 11 11.9
12 17.7
13 18.1
14 15.7
15 11.5
MV 15.0
SD 2.8

If not noted individually, the results include both lymph nodes of an animal. SD = standard deviation; MV = mean value

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, in a mouse local lymph node assay, the test item is considered to be a skin sensitizer.
Executive summary:

In a dermal sensitization study conducted according to OECD 429 with OO-tert-butyl monoperoxymaleate (47.3 % peroxide content) dissolved in AOO (4:1 (v/v) acetone / olive oil), young adult female CBA/CaOlaHsd mice (5 per dose group) were tested at concentrations of 3.125 % (v/v), 6.25 % (v/v) and 12.5 % (v/v) in a local lymph node assay (LLNA). Due to animal welfare reasons the negative control was shared and a periodically performed positive control (1% Phenlyenediamine in AOO) was used. The animals treated with the test item at concentration of 6.25% and 12.5% showed erythema grade 1 on day 3 which is not considered a sign of excessive irritation. There were no significant clinical observations in any of the animals treated with the test item at a concentration of 3.125% or the negative control. Neither mortality nor any effects on body weights were observed in any of the animals. Each of the three tested concentrations exceeded the stimulation index of 3 (9.5 (3.125%), 10.5 (6.25%), 15.0 (12.5%)). Therefore, an EC3 value could not be calculated. In this study, OO-tert-butyl monoperoxymaleate is a dermal sensitizer.