Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Endpoint summary

Description of key information

The substance was sensitising in a mouse local lymph node assay.

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:

05 August 2015 to 14 September 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species: Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA). Source: Janvier, Le Genest-Saint-Isle, France Number of animals: 20 females (nulliparous and non-pregnant), five females per group (main study only). Age and body weight: Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean. Identification: Tail mark with marker pen. Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality. Reliability check: The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed at half year intervals during at least the past 9 years showing reproducible and consistent positive results.Animal husbandry Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod is between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study. Accommodation: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). 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. Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Water: Free access to tap water. Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25% and 50%

No. of animals per dose:

Five females per group (main study)

Details on study design:

Test substance preparation Vehicle: Acetone/Olive oil (4:1 v/v) (Acetone p.a.: Merck, Darmstadt, Germany; Olive oil: Fagron, Nieuwerkerk a/d IJssel, The Netherlands). Rationale: The vehicle was selected on the basis of maximizing the solubility using the test substance data provided by the Sponsor and trial preparation results performed at WIL Research Europe. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol and dimethylsulfoxide. There was no information available regarding the solubility or stability in vehicle. Preparation: The test substance preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test substance is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed. Weight of evidence analysis In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the Sponsor, physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected. Pre-screen test A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied. Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (50% for solids). The test system, procedures and techniques were identical as those used in the main study except that the animals were approximately 12 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. 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 (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Since test substance remnants were to hamper ear thickness measurements on Day 3 for the animals treated at 50%, the ears were cleaned of residual test substance with the selected vehicle on Day 3 between 30 and 60 minutes prior to any further assessment. Scoring of the ears was conducted prior to dosing on Day 3. Ear thickness measurements on Days 3 were conducted following the scoring of irritation and prior to dosing. 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, 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 five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of 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: Following excision of the nodes, 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 then 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. Radioactivity 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).Observations Mortality/Viability: Twice daily. Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy). Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing). Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. In addition, a description of all other (local) effects was recorded.Necropsy: No necropsy for gross macroscopic examination was performed according to protocol.

Positive control substance(s):

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

Statistics:

None specified in the study report.

Key result

Parameter:

SI

Value:

> 3.2 - < 10.9

Parameter:

other: disintegrations per minute (DPM)

Value:

> 1 562 - < 5 300

Cellular proliferation data / Observations:

Pre-screen test Very slight erythema was noted for the animals treated at 50% on Day 3. Dark brown staining of test substance remnants on the dorsal surface of the ears did not hamper scoring for erythema but did hamper ear thickness measurements on Day 3 therefore the ears were cleaned with vehicle on Day 3. Bald spots behind the ears were noted for both animals treated at 50% from Day 3 onwards. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. No signs of systemic toxicity were observed in any of the pre-screen animals. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration. Main study Skin reactions / Irritation: No irritation and no signs of systemic toxicity were observed in any of the animals. Dark brown staining of test substance remnants on the dorsal surface of the ears did not hamper scoring for erythema. Scaliness and/or scabs were noted for three animals treated at 50% on Day 6. Bald spots behind the ears were noted for all animals treated at 50% from Day 2 onwards. Systemic toxicity: 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 experimental animals remained in the same range as controls over the study period. Macroscopic examination of the auricular lymph nodes and surrounding area: The auricular lymph nodes of one animal treated at 25% and four animals treated at 50% appeared enlarged in size. The auricular lymph nodes of the remaining animals were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Radioactivity measurements and SI values: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1562, 2207 and 5300 DPM, respectively. The mean DPM/animal value for the vehicle control group was 487 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 3.2, 4.5 and 10.9, respectively.

PRE-SCREEN TEST - Body weights and skin reactions

TS^1(%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw (g)^2

Erythema^3

Erythema

Erythema

Erythema

Erythema

Erythema

bw (g)

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

25

1 2

22.3 21.2

0F 0F

0F 0F

0F 0F

0F 0F

0F 0F

0F 0F

0 0

0 0

0 0

0 0

0 0

0 0

22.8 22.3

50

3 4

24.5 22.4

0F 0F

0F 0F

NS NS

NS NS

1 1

1 1

0F 0F

0F 0F

0F 0F

0F 0F

0SF 0SF

0SF 0SF

24.6 22.3

¹TS = test substance (% w/w)

²Body weight (grams)

³Grading erythema and eschar formation (Left = dorsal surface of left ear; Right = dorsal surface of right ear):

0 = No erythema

1 = Very slight erythema (barely perceptible)

NS = Dark brown staining of test substance remnants on the dorsal surface of the ears did hamper scoring of erythema

F = Dark brown staining of test substance remnants on the dorsal surface of the ears did not hamper scoring of erythema

S = Scaliness

 

PRE-SCREEN TEST - Ear thickness measurements

TS^1(%)	Animal	Day 1		Day 3				Day 6
		Left (mm)	Right (mm)	Left		Right		Left		Right
				(mm)	%^2	(mm)	%^2	(mm)	%^2	(mm)	%^2
25	1 2	0.225 0.225	0.225 0.225	0.245 0.245	9 9	0.245 0.245	9 9	0.240 0.225	7 0	0.225 0.225	0 0
50	3 4	0.220 0.220	0.225 0.220	0.235 0.250	7 14	0.245 0.255	9 16	0.240 0.235	9 7	0.240 0.235	7 7

Left (mm) = thickness of left ear in millimetres: Right (mm) = thickness of right ear in millimetres

¹TS = test substance (% w/w)

²Percent compared to Day 1 pre-dose value

 

MAIN STUDY - Body weights and skin reactions

Group	TS^1(%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			bw (g)^2	Erythema^3		Erythema		Erythema		Erythema		Erythema		Erythema		bw (g)
				Left	Right	Left	Right	Left	Right	Left	Right	Left	Right	Left	Right
1	0	1 2 3 4 5	23.3 22.3 21.3 22.1 21.9	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	23.8 23.2 21.6 21.9 21.0
2	10	6 7 8 9 10	23.6 22.5 21.2 23.7 23.0	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	22.3 24.6 22.2 22.3 22.2
3	25	11 12 13 14 15	21.3 25.1 20.7 21.9 22.5	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	22.2 25.0 21.3 23.1 23.4
4	50	16 17 18 19 20	22.5 19.6 23.9 24.2 23.7	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0F 0F 0F 0F 0F	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0FK 0K 0FK 0F 0F	0FK 0FS 0F 0F 0F	23.5 21.4 25.2 24.8 23.9

¹TS = test substance (% w/w)

²Body weight (grams)

³Grading erythema and eschar formation (Left = dorsak surface of left ear; Right = dorsal surface of right ear):

0 = No erythema

F = Dark brown staining of test substance remnants on the dorsal surface of the ears did not hamper scoring for erythema

S = Scaliness

K = Scabs

Note: Bald spots behind the ears were noted for all animals treated at 50% from Day 2 onwards.

 

MAIN STUDY – 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 2 3 4 5	N N N N N	N N N N N	289 493 739 275 638	487 ± 92	1.0    0.3
2	10	6 7 8 9 10	N N N N N	N N N N N	2064 1152 1683 952 1961	1562 ± 220	3.2 ± 0.8
3	25	11 12 13 14 15	N N N + N	N N N + N	2302 2505 3008 1559 1660	2207 ± 270	4.5 ± 1.0
4	50	16 17 18 19 20	N + + + +	N + + + +	807 2254 9947 6114 7376	5300 ± 1674	10.9 ± 4.0

¹TS = test substance (% w/w)

²Relative size auricular lymp nodes (-, -- or ---: degree of reductions, +, ++ or +++: degree of enlargement, N: considered to be normal)

³DPM = Disintegrations per minutes

⁴SEM = Standard Error of the Mean

 

SUMMARY RELIABILITY CHECK

Group^1	% Alpha-Hexylcinnamaldehyde, technical grade	Mean DPM ± SEM	SI ± SEM
1 2 3 4	0% (Acetone:Olive oil (4:1 v/v)) 5% 10% 25%	468 ± 91 808 ± 265 1391 ± 473 4243 ± 281	1.0 ± 0.3 1.7 ± 0.7 3.0 ± 1.2 9.1 ± 1.9

¹Five females per group

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine would be regarded as skin sensitizer.

Executive summary:

Assessment of skin sensitization to Formaldehyde, oligomeric reaction products with acetone and diphenylamine in the Mouse (Local Lymph Node Assay).

 

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2010),

EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

 

Test substance concentrations selected for the main study were based on the results of a pre-screen test.

 

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% 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.

 

No irritation and no signs of systemic toxicity were observed in any of the animals. Scaliness and/or scabs were noted for three animals treated at 50% on Day 6. Bald spots behind the ears were noted for all animals treated at 50% from Day 2 onwards.

 

The auricular lymph nodes of one animal treated at 25% and four animals treated at 50% appeared enlarged in size. The auricular lymph nodes of the remaining animals were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1562, 2207 and 5300 DPM, respectively. The mean DPM/animal value for the vehicle control group was 487 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 3.2, 4.5 and 10.9, respectively.

 

These results show that the test substance elicits a SI ≥ 3. The data showed a dose-response and the EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between >0 and 10%.

 

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

 

Based on these results:

- according to the recommendations made in the test guidelines (including all amendments), Formaldehyde, oligomeric reaction products with acetone and diphenylamine would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), Formaldehyde, oligomeric reaction products with acetone and diphenylamine should be classified as skin sensitizer (Category 1).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), Formaldehyde, oligomeric reaction products with acetone and diphenylamine should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

An assessment of the skin sensitising potential of formaldehyde, oligomeric reaction products with acetone and diphenylamine was conducted in the mouse (Local Lymph Node Assay).

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% 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.

No irritation and no signs of systemic toxicity were observed in any of the animals. Scaliness and/or scabs were noted for three animals treated at 50% on Day 6. Bald spots behind the ears were noted for all animals treated at 50% from Day 2 onwards.

The auricular lymph nodes of one animal treated at 25% and four animals treated at 50% appeared enlarged in size. The auricular lymph nodes of the remaining animals were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1562, 2207 and 5300 DPM, respectively. The mean DPM/animal value for the vehicle control group was 487 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 3.2, 4.5 and 10.9, respectively. These results show that the test substance elicits a SI ≥ 3. The data showed a dose-response and the EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between >0 and 10%.

Based on these results:, according to the recommendations made in the test guidelines (including all amendments), formaldehyde, oligomeric reaction products with acetone and diphenylamine is classified as skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin Sensitisation

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), formaldehyde, oligomeric reaction products with acetone and diphenylamine should be classified as skin sensitizer (Category 1) and labelled as H317: May cause an allergic skin reaction.