Carbonic acid, C,C'-[4-(2-bromoacetyl)-1,3-phenylene] C,C'-dimethyl ester

Endpoint summary

Description of key information

The dermal sensitisation potential of 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone was assessed in accordance with OECD guideline 429 ("Skin Sensitisation: Local Lymph Node Assay").

Under the conditions of the study 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone showed sensitising potential.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, The Netherlands
- Age at study initiation: 8 -12 weeks
- Weight at study initiation: 18.8 - 21.2 g
- Housing: group housing in Makrolon Type II (pre-test) and III (main study) with wire mesh top
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least five days prior to the start of dosing under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): The relative humidity in the animal room was between approximately 45 – 81.5 % instead of 45 – 65% for approx. 9 hours.
This deviation to the study plan, however, does not affect the validity of the study, as a temporary alteration in relative humidity over several hours is not considered to have any influence on the test system and the test results obtained. Furthermore, according to Test Guideline OECD 429 a relative humidity of 30 – 70% (with values above 70% of a short duration during room cleaning procedures) is acceptable.
- Photoperiod (hrs dark / hrs light): artificial light from 6.00 a.m. - 6.00 p.m.

Vehicle:

dimethylformamide

Concentration:

Main Test: 0.05, 0.1 and 0.25 %
Pre-Test: 50, 25, 10, 5, 2.5, 1, 0.5, 0.25 and 0.1 %

No. of animals per dose:

Main Test: five animals per dose
Pre-Test: one animal per dose

Details on study design:

see "any other information on material and methods incl. tables"

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
The EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
The Dean-Dixon-Test and the Grubb’s test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw). An outlier (animal 20) was identified in both outlier tests. However since exclusion of the outlier value would not change the overall test result, the value in question was not excluded from calculations.
A statistical analysis was conducted on the ear weights to assess whether the difference was statistically significant between test item groups and negative control (vehicle) group. Furthermore, a statistical analysis was conducted on the ear thickness values to assess whether a statistically significant increase in ear thickness could be observed when comparing the values measured on day 1 prior to application with the values measured on days 3 or 6 in the respective test item groups or within the vehicle control group.
For all statistical calculations validated statistical program R Script DecisionTree_2.Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
However, both biological and statistical significance were considered together.

Positive control results:

Results of the last 10 psoitive control experiments see table 3 in "any other information on results incl. tables".

Key result

Parameter:

SI

Value:

ca. 2.55

Test group / Remarks:

0.05 %

Remarks on result:

other: In this study Stimulation Indices (S.I.) of 2.55, 7.88 and 14.03 were determined eith the test item at concentrations of 0.05, 0.1 and 0.25 % in DMF, respectively.

Key result

Parameter:

SI

Value:

ca. 7.88

Test group / Remarks:

0.1 %

Key result

Parameter:

SI

Value:

ca. 14.03

Test group / Remarks:

0.25 %

1) Calculation and Resulte of Individual Data

Table 1: Calculation and results of individual data.

Test item concentration			DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
%	Group no.	Animal no.
---	---	BG I	13	---	---
---	---	BG II	13	---	---
0	1	1	1384	1371.0	---
0	1	2	1097	1084.0	---
0	1	3	965	952.0	---
0	1	4	1014	1001.0	---
0	1	5	1922	1909.0	---
0.05	2	6	944	931.0	0.7
0.05	2	7	4062	4049.0	3.2
0.05	2	8	4710	4697.0	3.7
0.05	2	9	4210	4197.0	3.3
0.05	2	10	2242	2229.0	1.8
0.1	3	11	5378	5365.0	4.2
0.1	3	12	10919	10906.0	8.6
0.1	3	13	15515	15502.0	12.3
0.1	3	14	6085	6072.0	4.8
0.1	3	15	11967	11954.0	9.5
0.25	4	16	18974	18961.0	15.0
0.25	4	17	19769	19756.0	15.6
0.25	4	18	20882	20869.0	16.5
0.25	4	19	18245	18232.0	14.4
0.25	4	20	10797	10784.0	8.5

1    =  Control Group

2-4=  Test Group

^a)   =  values corrected for mean background value (BGI and BGII)

^b)    =  Stimulation Indices relative to the mean of the control group (Group 1)

2) Calculation of Stimulation Indices per Dose Group

Table 2: Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (DMF)	1263.4	395.7	1.00
0.05% 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone	3220.6	1584.7	2.55
0.1% 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone	9959.8	4237.2	7.88
0.25% 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone	17720.4	3999.1	14.03

^a)      Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

Calculation of the EC3 value:

	Test item concentration %	S.I.
Test Group 2	0.05 (a)	2.55 (b)
Test Group 3	0.1 (c)	7.88 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 0.054%

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

3) Viability / Mortality

No deaths occurred during the study period.

4) Clinical Signs

No signs of systemic toxicity were observed during the study period.From day 3 to 5, the animals treated witha test item concentration of0.25% showed a very slight erythema of the ear skin (Score 1). Animals treated with 0.05 and 0.1% test item concentration did not show any signs of local skin irritation.

5) Body weights

The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

6) Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy).A statistically significant increasein ear weight was observed in the high dose group in comparison to the values of the vehicle control group.

7) Ear Thickness

The measured ear thickness of all animals treated was recorded prior to the 1^stapplication, on study day 3 and prior to necropsy (day 6).A statistically significant increase in ear thickness was observed for day 6 in all tested dose groups.

8) Lymph Node Weights

The measured lymph node weights of all animals treated were recorded after sacrifice.A statistically significant increase in lymph node weight was observed in the mid and high dose group.

9) Historical Positive Control Data

Table 3: Historical control data of the last 10 positive control experiments

Positive Control Substance	Date	Concentration / Vehicle	S.I. values
alpha- hexyl-cinnamaldehyde	April 2015	25% in acetone:olive oil (4+1 v/v)	9.5
	October 2014		13.8
	April 2014		6.8
	October 2013		5.8
	April 2013		5.9
	October 2012		5.7
	April 2012		3.7
	February 2012		4.7
	January 2012		10.8
	January 2012		7.1

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item 2',4'-Bis(methoxycarbonyloxy)-2-brom-acetophenone was found to be a skin sensitiser under the conditions of this study.

Executive summary:

In a study according to OECD 429 ("Skin Sensitisation: Local Lymph Node Assay") 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone formulated in DMF was assessed for its possible skin sensitising potential. The test was performed in mice using test item concentrations of 0.05, 0.1 and 0.25 % (w/v). The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 3 to 5, the animals treated with a test item concentration of 0.25 % showed a very slight erythema of the ear skin (Score 1). Animals treated with 0.05 and 0.1 % test item concentration did not show any signs of local skin irritation. A statistically significant increase in ear thickness was observed for day 6 in all tested dose groups as well as in ear weiht in the high dose group, when compared to the vehicle control group. However increases in ear weights and/or ear thickness were below the threshold of >= 25 % increase as described in OECD 429.

In this study Stimulation Indices (S.I.) of 2.55, 7.88 and 14.03 were determined with the test item at concentrations of 0.05, 0.1 and 0.25 % in DMF, respectively. A clear dose response was observed. Furthermore, a statistically significant increase in lymph node weight was observed in the mid and high dose group, corroborating the presence of a positive response. The EC3 value calculated was 0.054 %. Therefore the test item 2',4'-Bis(methoxycarbonyloxy)-2-brom-acetophenone was found to be a skin sensitiser under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The dermal sensitisation potential of 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone was assessed in accordance with OECD guideline 429 ("Skin Sensitisation: Local Lymph Node Assay").

Under the conditions of the study 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone showed sensitising potential.

Justification for classification or non-classification

The dermal sensitisation potential of 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone was assessed in accordance with OECD guideline 429 ("Skin Sensitisation: Local Lymph Node Assay").

Under the conditions of the study 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone showed sensitising potential and was as a skin sensitising substance.

Therefore the substance 2',4'-Bis(methoxycarbonyloxy)-2-brom-acetophenone has to be classified as Skin. Sens. 1 with "H317: May cause an allergic skin reaction".