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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The GLP study was conducted according to an internationally accepted guideline. All study parameters are given in detail. Nevertheless, according to the ECHA's practical guide 6: "How to report read-across and categories" the maximum for read-cross is 2.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 18.1 - 22.6°C), a relative humidity of 40-70% (actual range: 46 - 68%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

Accommodation

Animals were group housed in labeled Makrolon cages (MI 11 type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment. Certificates of analysis were examined and then retained in the NOTOX archives.

Diet

Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany). Water

Free access to tap water.

Results of analysis for diet (nutrients and contaminants), sawdust, paper, shelters and water were assessed and did not reveal any findings that were considered to have affected the study integrity.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Group animal numbers induction (test substance; % w/w)

1 01 - 05 0 (Acetone/Olive oil (4:1 v/v))
2 06 - 10 5
3 11 - 15 10
4 16 - 20 25 

No. of animals per dose:
5 animals
Details on study design:
Pre-screen test
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at the most (maximum grade 2 (see section 6.7) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 100% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids).
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. The surviving animals were sacrificed after the final observation.

Main study
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test.
One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal)
with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 1.5 and 6.6 respectively.
An EC3 value of 14.4% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.8, 13.9, 16.0, 11.9, 16.9 and 10.7%.
Based on these results it was concluded that the Local Lymph Node Assay is suitable for testing for contact hypersensitivity.
Parameter:
SI
Remarks on result:
other: see below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see below

Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

group

TS(1)

animal

Size nodes(2)

DPM(3)/animal

Mean DPM ± SEM(4)

Mean

 SI±SEM

left

right

1

0

1

n

n

70

 

 

 

 

2

n

n

130

 

 

 

 

3

n

n

137

97±15

1.0±0.2

 

 

4

n

n

89

 

 

 

 

5

n

n

61

 

 

 

 

 

 

 

 

 

 

2

5

6

n

n

173

 

 

 

 

7

n

n

188

 

 

 

 

8

n

n

86

139±18

1.4±0.3

 

 

9

n

n

119

 

 

 

 

10

n

n

131

 

 

 

 

 

 

 

 

 

 

3

10

11

n

n

125

 

 

 

 

12

n

n

72

 

 

 

 

13

n

n

82

98±9

1.0±0.2

 

 

14

n

n

106

 

 

 

 

15

n

n

105

 

 

 

 

 

 

 

 

 

 

4

25

16

n

n

93

 

 

 

 

17

+

+

199

 

 

 

 

18

n

n

96

132±27

1.4±0.3

 

 

19

n

n

78

 

 

 

 

29

+

n

196

 

 

1 TS = test substance (% w/w).

2. Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

3. DPM = Disintegrations per minute

4. SEM = Standard Error of the Mean

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Phenyl-tolyl-ethane was considered not to be a skin sensitizer in the Mouse Local Lymph Node Assay according to OECD guideline 429.
Executive summary:

A study was performed to assess the Contact Hypersensitivity to Phenyl-tolyl-ethane in the Mouse (Local Lymph Node Assay). The study was carried out based on the guideline OECD, Section 4, Health Effects, No.429 (2010).

Test substance concentrations selected for the main study were based on the results of a pre-screen test. The two animals treated at 100% were found dead or sacrificed for humane reasons on Day 2. The two animals treated at 50% showed signs of systemic toxicity (hunched posture). It was concluded that these concentrations could not be tolerated well and did not comply with the selection criteria, and therefore the highest test substance concentration selected for the main study was a 25% concentration. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. The slight irritation of the ears as shown by the animals treated at 25% was considered not to have a toxicologically significant effect on the activity of the nodes. No irritation of the ears was observed in any of the animals treated with vehicle or at test substance concentration of 10% or 5%. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of the experimental animals remained in the same range as controls over the study period, except for the slight body weight loss noted in two animals treated at 25% and one at 5%. This body weight loss was considered not toxicologically significant. The auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for the enlarged nodes found in two animals treated at 25%. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 139, 98 and 132 DPM respectively. The mean DPM/animal value for the vehicle control group was 97 DPM. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 1.0 and 1.4 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to the highest tolerable concentration of 25%, Phenyl-tolyl-ethane was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%. Based on these results, Phenyl-tolyl-ethane would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Members of the diphenylmethane category did not show any skin sensitising properties. There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. For skin sensitisation, there is no reason to believe that results obtained in experimental animals would not be applicable to humans.

No further testing is required. The available data is adequate for risk assessment and classification and labelling purposes.


Migrated from Short description of key information:
Available information:
SAS-40:
A GLP-study was performed to assess the skin sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 "Skin Sensitisation" - Buehler Test. SAS-40 was found to be a non-sensitiser to guinea pig skin.

SAS-296: The skin sensitisation potential of SAS-296 was assessed in a non-GLP Bühler Test according to the method described in" Delayed Contact Hypersensitivity in the Guinea-pig" Buehler E.V. (1965), Arch. Dermatol. 91, 171.SAS-296 was found to be a non-sensitiser to guinea pig skin.


Phenyl-tolyl-ethane:
A study was performed to assess the Contact Hypersensitivity to Phenyl-tolyl-ethane in the Mouse (Local Lymph Node Assay). The GLP-study was carried out based on the guideline OECD, Section 4, Health Effects, No.429 (2010). Phenyl-tolyl-ethane was found to be a non-sensitiser.

Justification for selection of skin sensitisation endpoint:
3 GLP-studies according to internationally accepted guidelines are available. Negative test data from tested members of the diphenylmethane category can be extrapolated to the untested substance 1,1’-DPE.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

1,1 -DPE is not classified as skin sensitiser.