2-ethylhexyl acrylate

Carcinogenicity

Description of key information

The carcinogenicity data of 2-EHA have been extensively reviewed by the German Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (BAuA) in the frame of the EU risk assessment (2005), The conclusion of this assessment is reported below:  

"The carcinogenic potential of 2-EHA was tested in skin painting studies on the shaved back skin of male mice of different strains. None of the studies was performed according to the current regulatory recommendations on the EEC methods B 32 or B 33. The main defaults were that exclusively male mice were tested and that the effects on internal organs were not or insufficiently examined and documented resulting in an incomplete database on carcinogenic activities after absorption. 

2-EHA induced skin irritation and in nearly half the male C3H/HeJ mice treated dermally with 2-EHA solutions > 21% benign and malignant skin tumours (Wenzel-Hartung et al., 1989; Brune and Deutsch-Wenzel, 1986). Tumour incidence showed no relation to the dosage group of 21% or 86.5%. The additional study where treatment of 43% of 2-EHA were stopped at week 24 did not reveal any skin tumour. The weight of evidence that the test substance is carcinogenic is limited by the occurrence of skin irritation assumed to represent the precursor lesion of tumour growth. Repeated regenerative or proliferative reactions to irritative substances are discussed to be strongly associated to tumour development (Hasegawa et al., 1989). Even chronic physical stimuli such as abrasion in untreated skin were demonstrated to play a role in skin tumour induction. In general, dermal carcinogenicity studies should use test substance concentrations which did not induce irritative effects. In the BASF study (1986), no tumours were seen in the low dose group applicating 2.5% 2-EHA, although transient mild skin irritation was seen up to the 11th week of treatment. Contrary to positive cancer studies, skin irritation but no skin tumour was found in male NMRI mice treated dermally with 21.5%, 43% or 85% of 2-EHA in acetone for 24 months (BASF, 1992). 

Higher incidences of skin tumours were also evident in two other dermal studies in mice. However in first study only one dosage (75% 2-EHA in acetone) was tested in C3H/HeJ mice where the mortality of unknown cause was markedly increased (94-97) already at one year of the treatment period. No clear tumour response was demonstrated in the two-stage carcinogenicity model using 2-EHA as the initiator substance and TPA as promoter in the study on NMRI mice (BASF, 1992). The negative response in this study may be related to the different strain used in this study (NMRI mouse) compared to the C3H/HeJ80 mouse of earlier studies that were positive. One skin tumour bearing animal occurred at each dose after 7 months treatment to 2-EHA and an additional treatment period to the promoter TPA. 2-EHA as well as TPA was shown to be irritative to the skin. Both studies were considered to be inadequate to detect the presence of carcinogenic effects.

Skin tumours from spontaneous origin are known to be variable in different mouse strains. One out of 41 control animals of the study of BASF (1992) had a squamous cell papilloma at an untreated skin area. No other spontaneous skin tumour was reported in the control groups of treated and untreated skin areas of the above cited studies. 

From oral (the only study with validity according to the cancerogenicity test guidelines) and dermal studies on acrylic acid, the hydrolysis product of 2-EHA, there is no evidence on carcinogenic properties. Also, there is no concern from cancer data on 2-ethylhexanol. 

In conclusion, there are no data available to the carcinogenic effects with respect to oral or inhalative exposure routes.

Findings from the dermal mouse carcinogenicity study showed that 2-EHA induces skin tumours at concentrations which were highly irritative. It was concluded, that tumour growth is associated the highly irritative properties of 2-EHA. At a low concentration of 2.5% 2-EHA with transient irritation no tumour response of the skin was observed. Other long-term studies on different mouse strains did not confirm tumour induction of the mouse skin. Additionally, there is no concern from tumour data of acrylic acid and 2-ethylhexanol, the hydrolysis products of 2-EHA.

Taking into account the negative results from in-vivo genotoxicity testing, it is concluded that 2-EHA induces skin tumours by a non-genotoxic mechanisms. Irritative skin damage was identified as presumed mode of tumourigenicity as to was associated with carcinogenic effect of 2-EHA. Due to the limited reliability of skin painting studies in mice as a tool to identify the carcinogenic potential of a test substance these studies give some concern but no clear evidence that 2-EHA has carcinogenic potential. Based on limited database from dermal studies and absence of carcinogenicity data for the oral and inhalation routes, no conclusion could be drawn about the carcinogenic potential of 2-EHA. However taking into account the negative experimental results from long term animal studies with the cleavage product acrylic acid after oral and dermal application (see EU Risk Assessment Report Acrylic acid) there are no reasons to assume that 2-EHA should be considered as a carcinogenic substance."

In additon based on recent publications the skin painting studies in C3H/HeJ mice using 2EHA have to be regarded as non-reliable. The C3H/HeJ mouse model is not appropriate as it has a mutation in Toll-like receptor 4 (TLR4) that impairs its innate and adaptive immune

responses. Inconsistencies in the histological evaluation of tumors induced in C3H/HeJ mice provide further evidence that the tumorigenic effect of 2-EHA was strain specific, a result of chronic inflammation during the promotion stage and/or a skewed immune response caused by the TLR4 mutation. (Elmets and Yusuf, Biomed Hub 2020;5:508295; Murphy et al. 2018, Toxicology Letters)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June 1987 - 28 June 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, conducted in compliance with GLP regulations

Principles of method if other than guideline:

Skin painting study

GLP compliance:

yes

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl : NMRI BR
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 48 to 50 days old
- Mean weight at study initiation: 34.5 (29.8 - 39.6) g
- Housing: single
- Diet (ad libitum): Kliba rats/mice/hamsters maintenance diet, "A" 343 pellets, Klingentalmuehle AG, CH-4303 Kaiseraugst, Switzerland
- Water (ad libitum): drinking water
- Acclimation period: 9 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 709K
- Photoperiod (hrs dark / hrs light): 12 h dark/ 12 h light

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

ADMINISTRATION
- Route of administration: epicutaneously to the clipped interscapular region by dropping 25 ul of the respective 2-EHA concentration dissolved in acetone per mouse per application using a Hamilton Digital Diluter.

TEST SITE
- Area of exposure: interscapular region
- Type of wrap if used: no

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 uL
- Concentration (if solution): 21.5; 43.0; 85.0 % (w/w) in acetone
- Constant volume used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of 2-ethylhexyl acrylate in acetone for a period of 4 weeks was verified analytically before the start of the study under ZST No. 80/429 (Analytical Report, Inhalation ZST-E, 04 Dec 1985).
The analytical determinations of the concentrations of 2-ethylhexyl acrylate in acetone confirmed that the concentrations were correct.

Duration of treatment / exposure:

24 months

Frequency of treatment:

three times per week (Mondays, Wednesdays and Fridays)

Post exposure period:

none

Dose / conc.:

21.5 other: % (nominal)

Remarks:

269 mg/kg/d

Dose / conc.:

43 other: % (nominal)

Remarks:

538 mg/kg/d

Dose / conc.:

85 other: % (nominal)

Remarks:

1063 mg/kg/d

No. of animals per sex per dose:

80 in the main study

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:

The doses were chosen on the basis of the following investigations:
1) Dermal oncogenicity study of 2-ethylhexyl acrylate by chronic epicutaneous application in male C3H mice. Beratungsforum fuer Praeventivmedizin und Umweltschutz GmbH (Advisory Forum for Preventive Medicine and Environmental Protection); 20 Nov 1986.
2) Comparative study of dermatologic effects of 2-ethylhexyl acrylate on 2 mice strains after 3-month epicutaneous application. Beratungsforum fuer Praeventivmedizin und Umweltschutz GmbH (Advisory Forum for Preventive Medicine and Environmental Protection).

The dose levels for the study were fixed based upon the doses of the carcinogenicity study on C3H mice mentioned under 1).

In that study, 86.5 %, 21 % and 2.5 % 2-ethylhexyl acrylate solutions were administered throughout the life-span of the animals and a 43 % solution was epicutaneously administered for 26 weeks ("Stop test").
The application of 86.5 % and 21 % 2-Ethylhexyl acrylate resulted in the formation of skin tumors and chronic dermal inflammation. In the stop test performed at a concentration of 43 %, the inflammatory skin changes receded in the observation period and no tumors were observed. No notable dermal inflammation occurred after life-long application of 2.5 % 2-ethylhexyl acrylate.
Taking the results of a comparative 3-month study with 2-ethylhexyl acrylate between C3H and NMRI mice (2) as a point of departure where, as regards the irritation challenged by 2-ethylhexyl acrylate, the C3H mouse was more sensitive than the NMRI mouse, the question was to clarify if 2-ethylhexyl acrylate also exhibits a carcinogenic potential on the skin of NMRI mice or if the tumors found in the C3H mouse are to be assessed as a reaction specific for this strain.

Dosage groups:
0) solvent control (acetone)
1) positive control (0.015 % w/w Benzo(a)pyrene)
2) 21.5 % 2-EHA (w/w)
3) 43 % 2-EHA (w/w) (stop of treatment: 24th week); observed life-span (stop test)
4) 85 % 2-EHA (w/w)


- Rationale for selecting satellite groups:

In order to examine early changes in the skin after about 3 months of administration or to detect a possible initiating capacity of the test substance, respectively, the test groups were divided during the study into several groups with a differing treatment schedule:

Interim:
Two animals from each group were sacrificed after about 3 months of the study for further information on the degree of severity of the clinical findings on the skin.

After about 7 months of treatment, the test groups 0-4 were divided into equal halfs and further treatment was performed according to the following schedule:

Main 1:
After a treatment-free period of five days, 41 (group 0), 40 (group 2), and 39 animals (groups 1,3,4) were treated as scheduled for 24 months and then sacrificed.
Main 2:
After a treatment-free period of about 2 months, 37 (group 0P), 36 (groups 1P, 4P), 30 (group 2P), and 39 (group 3P) animals were treated for about 20 weeks with the tumor promotor 12-0-tetradecanoylphorbol-13-acetate (TPA) (2x/week, 5 ug in 0.1 mL acetone) and then kept without treatment for up to 24 months.
Animals of test groups 0P - 4P that were not treated with 12-0-tetradecanoylphorbol-13-acetate due to humane reasons were allocated to test groups 16 - 20.


- Post-exposure recovery period in satellite groups: 24 months (groups 0P - 4P)

Positive control:

benz(a)pyrene

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made for dead or moribund animals twice a day (Monday to Friday) or at least once a day (Saturdays, Sundays and public holidays). The general state of health was checked at least daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were additionally inspected and palpated once a week, including an exact inspection of the skin.


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: once a week.


BODY WEIGHT: Yes
- Time schedule for examinations: One day before the start of the study (day 0) the body weight was determined in order to randomize the animals. The body weight of the animals was determined once a week until the 14th week and then in 4-weekly intervals.

Sacrifice and pathology:

At the end of the study, all surviving animals were sacrificed and submitted to necropsy and gross-pathological examination. Subsequently the following organs or tissues were fixed in 4 % formaldehyde solution:
- skin of the interscapular region (mounted on a plate)
- animal body with all organs and all grossly visible lesions (undissected). The skin of the interscapular region of all animals was examined under light-microscopy. Other skin sites which showed lesions were also examined microscopically.


GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Statistical significance of the clinical data (body weight) were determined by analysis of variance (ANOVA) followed by a DUNNETT's test (1955, 1964).

DUNNETT CW (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Statist. Assoc. 50: 1096 - 1121.
DUNNETT CW (1964). New Tables for multiple comparisons with a control. Biometrics 20: 482 - 491.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

INTERIM SACRIFICE
The application of 2-ethylhexyl acrylate (2-EHA) in concentrations of 21.5 %, 43 %, or 85 % over a period of 3 months (interim) revealed only signs of mild dermal irritation with no clear dose-response relationship.


MAIN 1 (WITHOUT PROMOTOR)
After an application period of 24 months (test groups 2-4), in principle the same observations as seen after 3 months of treatment had persisted in the treated skin, some of which exhibited relationship to the applied concentration (like hyperkeratosis, crust formation, ulceration), other without a clear relationship to the concentrations applied (like hyperplasia, lymphocyte infiltration, increased macrophages, dermal fibrosis).

SOLVENT AND POSITIVE CONTROLS
In the acetone-treated control group (test group 0), only few animals were seen with clinical symptoms in the treated skin, and in the benzo(a)pyrene (BaP) treated group (test group 1) skin lesions were seen resulting usually from ulceration of existing tumors.


MAIN 2 (WITH PROMOTOR)
In all groups being treated with 12-0-tetradecanoylphorbol-13-acetate (TPA) during the promotion phase (test groups 0P to 4P), signs of dermal irritation like hyperkeratosis, hyperplasia, crust formation or ulceration were observed comparably often in animals pretreated with either acetone, 2-EHA, or BaP underlining the irritating potential of TPA itself. Neither 2-EHA or TPA caused clinical signs beside skin changes, which could be causally related to their administration. However, in the BaP-treated positive control groups all animals either died prematurely or had to be sacrificed in a moribund state.


HISTOPATHOLOGY: NEOPLASTIC
As shown in the table (see below), most of the masses being observed clinically throughout the study could also be seen at necropsy. The difference between the number of masses observed clinically and the number of masses observed at necropsy was related to the regression, loss or ulceration of the pediculate growing masses. With one exception (one animal of test group 1), all masses observed at necropsy were confirmed histopathologically as being squamous cell carcinomas, squamous cell papillomas, or keratoacanthomas. In test groups 0 to 4, malignant tumors in the treated skin (i.e. squamous cell carcinomas) were observed only in the positive control group (benzo(a)pyrene, test group 1) in 31 out of 39 animals. In two other animals of this group as well as in 3 animals of test group 17 a benign squamous cell papilloma was diagnosed in the treated skin. After promotion with TPA, malignant skin tumors (squamous cell carcinomas) and benign keratoacanthomas occurred only in the animals of the benzo(a)pyrene group 1P (24 and 5 out of 36 animals, respectively). A single squamous cell papilloma was induced in any of the treatment groups (groups 2P to 4P) and in the benzo(a)pyrene group (group 1P), but not in the concurrent control group (group 0P).



The development of these tumors is concluded not to be related to the treatment of the skin with 2-EHA
- as a squamous cell papilloma occurred spontaneously in the untreated skin of the concurrent control group 0P, underlining the possibility of the spontaneous occurrence of this type of a tumor;
- no clear indication of a dose-response relationship to the applied concentrations of 2-EHA was evident;
- the induction of the tumors might be connected with, if not even related, to the irritating potency of TPA itself;
- in a previous study where 2.5 ug TPA were administered to the skin three times weekly for 20 weeks to 40 female Swiss ICR-mice, a squamous cell papilloma and a keratoacanthoma were observed in the treated skin of the control animals after only 52 weeks. In addition, severe dermal irritation (hyperplasia, erosions and/or ulcers) were observed (BASF AG, Project No. 93C0029/87009).


CONCLUSION
Thus, under the test conditions chosen, it can be concluded that the administration of 2-ethylhexyl acrylate to the skin of male NMRI mice at doses of 21.5 % , 43 %, and 85 % (w/w) for two years did not result in the formation of tumors in the treated skin. The same dosing regimen administered for about 7 months, followed by treatment with 12-0-tetradecanoylphorbol-13- acetate for 20 weeks also resulted in findings which were assessed as giving no indication for a carcinogenic potential of 2-ethylhexyl acrylate on the skin of male NMRI mice.

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

85 other: % (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Dose descriptor:

LOAEL

Remarks:

local nonneoplastic skin effects

Effect level:

21.5 other: % (nominal=

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Histopathological findings:

Concerning neoplastic findings in the treated skin, a summary of the masses being observed clinically or at necropsy as well as a summary of the histologically confirmed tumors is given in the following table:

Group	Treatment	No. of		No. of		No. of		Histologically confirmed tumors:
			animals		animals		animals		
					with masses	with masses	squamous	squamous	kerato-
					being		being		cell		cell		acanthoma
					observed	observed	papilloma	carcinoma
					clinically	at necropsy
----------------------------------------------------------------------------------------------------------------------
0	Acetone		41		0		0		0		0		0
1	B(a)P		39		32		27		2		31		0
2	21.5 % TS	40		0		0		0		0		0
3	43.0 % TS	39		0		1		0		0		0
4	85.0 % TS	39		0		0		0		0		0

0P	Acetone + TPA	37		0		0		0		0		0
1P	B(a)P + TPA	36		30		25		1		24		5
2P	21.5 % TS + TPA	30		1		1		1		0		0
3P	43.0 % TS + TPA	39		2		1		1		0		0
4P	85.0 % TS + TPA	36		1		0		1		0		0

11	Acetone, 13w	2		0		0		0		0		0
12	B(a)P, 13w	2		0		0		0		0		0
13	21.5 % TS, 13w	2		0		0		0		0		0
14	43.0 % TS, 13w	2		0		0		0		0		0
15	85.0 % TS, 13w	2		0		0		0		0		0

16	Acetone		0		0		0		0		0		0
17	B(a)P		3		3		3		3		0		0
18	21.5 % TS	8		0		0		0		0		0
19	43.0 % TS	0		0		0		0		0		0
20	85.0 % TS	3		0		0		0		0		0
----------------------------------------------------------------------------------------------------------------------
TS: 2-Ethylhexyl acrylate (test substance)
B(a)P: benzo(a)pyrene
TPA: 12-0-tetradecanoylphorbol-13-acetate

Executive summary:

2-EHA was tested for carcinogenicity in male NMRI mice exposed dermally 3 times/week to 25 µl of 21.5%, 43%, or 85% 2-EHA (w/w) diluted in acetone (approximately 269, 538 and 1,063 mg/kg bw/day). 39-40 of 80 males tested were treated with 2-EHA alone, acetone (solvent control) or 0.015% (w/w) of benzo(a)pyrene in acetone (positive control, results were not reported here) for up to 24 months (BASF, 1992). Two animals of each group were killed at week 13 to examine the skin lesions. Nearly half of the animals (30-39 males) of each dose and control groups were treated as above for 7 months, after a treatment-free period of two months animals were treated with a promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 20 weeks. Thereafter there was no further treatment until the end of the study. Histopathology data were reported on the treated skin of all test animals and on the untreated skin area of 5-10 animals per group. Other organs/tissues were not included in histopathology examinations.

Whereas premature deaths were seen in the benzo(a)pyrene control group no treatment-related effect on mortality was observed in the 2-EHA groups. Neither 2-EHA nor the promoter TPA caused clinical signs besides the skin effects. Treatment-related lesions of the treated skin region were observed after 13 weeks and after 24 months in all 2-EHA groups. Some findings as hyperkeratosis, hyperplasia, crust formation and ulceration increased in severity or incidence related to the doses of 2-EHA, others (lymphocyte/macrophage infiltration, dermal fibrosis) were observed without any relation to the treatment groups. Reddening and thickening of the skin and similar microscopic skin lesions were reported during the promoter-phase. The authors concluded an irritative effect of the promoter TPA itself.

6/41 animals of the acetone group showed mild clinical symptoms (hyperkeratosis, hyperplasia, ulceration or lymphocytic/macrophage infiltrations).

None of animals treated with 2-EHA for up to 24 months showed a neoplastic lesion of the skin. 2-EHA and TPA promotion caused a squamous cell papilloma in one animal in each dose groups (of 30, 39, and 36 animals). One squamous cell papilloma was found in untreated skin of one male out of 41 of the acetone group.

The authors concluded that these tumours were related to the irritative effects of TPA, not to the treatment with 2-EHA.The LOAEL for nonneoplastic toxic effects of 2-EHA on the skin was 25 µl 2-EHA solution at a concentration of 21.5% 2-EHA in acetone (269 mg/kg bw/day).

The findings did not indicate a carcinogenic potential of 2-EHA on the skin of male NMRI mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 063 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Mode of Action Analysis / Human Relevance Framework

In an expert review (Elmets and Yusuf, 2020) of the carcinogenicity studies performed on 2 -EHA and of the underlying mechanism was evaluated and it was concluded:

“While not to deny the value of these reports, it is important to recognize their limitations.

1) Very high doses/concentrations of 2-EHA were employed substantially greater than that to which humans are exposed. In addition, this monomer is only handled in workplaces under controlled conditions that minimize or eliminate exposure.

2) At all of the doses used, topical application of 2-EHA caused crusting, inflammation and ulcerations of the skin.

3) C3H/HeJ mice are an imperfect model for cancer assessments because they have deviant innate and adaptive immunity due to an inactivating mutation in the gene that encodes the TLR4 gene.

4) Skin lesions regressed after application of 2-EHA (43%) was discontinued. No tumors could be seen in the low dose (2.5%) group when skin irritation occurred only during the initial phases of the experiment. These observations provide strong evidence that chronic inflammation and immune dysregulation, rather than genotoxic injury was responsible for tumors that occurred (Wenzel-Hartung et al., 1989).

5) The MMTV status of the C3H/HeJ mice was not stated; if these mice carried the virus, it may have influenced the results of the skin carcinogenesis studies.

The types of tumors that developed in C3H/HeJ mice are noteworthy. Premalignant papillomas, squamous cell carcinomas, fibrosarcomas, basal cell carcinomas and melanomas were reported. The pathogeneses of these neoplasms are all different, with distinct mutations in each type. This provides additional support for the concept that the effect of 2-EHA in that strain was caused by a deviant immune response to tumor antigens caused by the TLR4 mutation. The fact that melanomas arose in 2-EHA treated mice is surprising since there were no good models of melanomagenesis until recently (Nasti et al., 2016). Thus, it is questionable as to whether they were actually tumors; it is likely that they were melanophages that had phagocytosed melanin in the dermis. There had also been no good murine models of basal cell carcinoma at the time that the study was conducted. Current methods that are used to definitively identify melanomas include immunohistochemical and/or mRNA evaluation of lesions for tyrosinase, S100, and MelanA as well as examination for compatible genetic mutations (Nasti et al., 2016).”

 

In addition, Murphy et al., (2018) highlighted that the dose selection for the DePass, (1985) screening study in C3H/HeJ mice was based on a 2-week study and tissue examination was limited to a gross evaluation. According to contemporary criteria (OECD, 2012; USEPA, 1988; USEPA, 1998) this is insu¿cient for proper maximum tolerated dose (MTD) de¿nition. A subchronic dermal study was later conducted to assess the irritant properties of 2-EHA solutions in C3H/HeJ mice as a range ¿nder for the chronic studies. In the C3H/HeJ chronic study (Brune and Deutsch-Wenzel, 1986; Wenzel-Hartung et al., 1989), 12 weeks of treatment produced eschar in 67 (84%), 49 (61%) and 52 (65%) of mice administered 86.5%, 43% and 21% 2-EHA, respectively, clearly exceeding the MTD and prompting the addition of a 2.5% group. Therefore, MTD for dermal application was clearly exceeded at dose levels >21% in C3H/HeJ mice. 

 

Using current guidance for setting the MTD, the 2.5% dose in the C3H/HeJ mouse met the criteria for the MTD, and no skin tumors developed at this dose. The dose ranges in the C3H/HeJ studies exceeded the MTD for skin integrity based on interim and terminal evaluations of the application site skin condition. Nonetheless, even studies with design ¿aws can provide useful information related to hazard (Rhomberg et al., 2007), however tumor response information in excess of an MTD should not be considered in a human hazard or risk assessment paradigm. For the purposes of an appropriate hazard assessment, 2-EHA did not cause or initiate dermal carcinogenesis in an immune competent (NMRI) mouse model (BASF AG, 1992), and, even in an immune compromised (C3H/HeJ) mouse model, did not induce skin tumors at doses which did not exceed the MTD.

 

Less major methodological deficiencies also exist in the DePass et al., 1985

 

Additional references:

Elmets and Yusuf (2020, Biomed Hub 2020;5:508295 attached in IUCLID section 7.7)

Murphy, S., Ellis-Hutchings, R., Finch, L., Welz, S., and Wiench, K. (2018). Critical evaluation of 2-ethylhexyl acrylate dermal carcinogenicity studies using contemporary criteria. Toxicology Letters, 294: 205-211 (attached in IUCLID section 7.7).

Nasti, T.H., Cochran, J.B., Tsuruta, Y., Yusuf, N., McKay, K.M., Athar, M., Timares, L., and Elmets, C.A. (2016). A murine model for the development of melanocytic nevi and their progression to melanoma. Mol Carcinog 55, 646-658.

OECD, 2012. Guidance document 116 on the conduct and design of chronic toxicity and carcinogenicity studies. Supporting Test Guidelines 451, 452 and 453, 2nd edition. 13 April 2012.

Rhomberg LR, Baetcke K, Blancato J et al. (2007)Issues in the Design and Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: Approaches to Dose Selection. Critical Reviews in Toxicology, 37(9):729-837.

USEPA, 1988. Summary of the Second EPA Workshop on Carcinogenesis Bioassay via the Dermal Route, May 18-19, 1988 Research Triangle Park NC. EPA560/6-89-003.

USEPA, 1998. Health E¿ects Test Guidelines Carcinogenicity OPPTS 870.4200. EPA 712–C–98–211.

Justification for classification or non-classification

GHS classification (GHS UN rev.4, 2011) identical to REGULATION (EC) No 1272/2008: no classification required

Additional information

 As reported in the EU RAR (2005): 

 

 There are dermal carcinogenicity studies in mice, but no cancer studies by oral or inhalation route. 

 

 In an early dermal life-time carcinogenicity study (DePass et al., 1985; DePass, 1982; Peterson, 1979; Slesinski et al., 1980) 40 male C3H/HeJ mice were treated 3 times/week with 2-EHA (75% w/v) dilution in acetone at an average dose of 20 µg 2-EHA/application (approximately 750 mg/kg bw/day). 6 of 40 treated males developed neoplastic skin lesions. Four males had squamous cell papillomas and two others had squamous cell carcinomas. One out of 40 animals of the vehicle control group developed a skin carcinoma near the eye. The authors concluded that 2-EHA is carcinogenic in C3H mice. 

 

 The study is less reliable due to the method defaults (see also the section “Detailed information on mode of action / Human relevance framework”). Only 40 males were investigated, exclusively gross lesions of skin and internal organs were examined histologically. Several tumours were documented grossly and most of them were examined histologically. Peterson (1979) reported several cases of chronic nephritis in 13 of 19 kidneys examined and necropurulent nephritis in several cases. Survival rate after one year of treatment was reduced (75%); the first skin tumour was seen at 11 months of treatment. At 18 months only 15/40 male and at 24 months none of them were still alive. As skin tumours were seen in a total of 6/40 mice, the cause of premature deaths remained unclear. Furthermore there were no data on irritative skin effects due to the treatment, histopathology of the skin were reported from seven males only. Tumours in non-cutaneous tissues were reported to be comparable between treated and control groups, however tumour data in internal organs were insufficient.

 

 The tumor response in the screening skin painting study in C3H/HeJ mice was unexpected, and prompted two more standard scale studies using C3H/HeJ (Brune and Deutsch-Wenzel, 1986; Wenzel-Hartung et al., 1989) and NMRI (BASF AG, 1992) mice. The outcome of the follow-up studies varied by mouse strain. 

 

Groups of 80 male C3H/HeJ mice received 3 applications per week of 25µ1 of 2-EHA solution (86.5 %, 21 %, or 2.5 % solution in acetone, approximately 937; 212 and 24.8 mg/kg bw/day) to the clipped dorsal skin over their lifetime (Brune and Deutsch-Wenzel, 1986; Wenzel-Hartung et al., 1989). Another group was treated with a 43 % 2-EHA solution (approx. 444 mg/kg bw/day); for 24 weeks and thereafter observed for lifetime (stop-test). An untreated group and acetone group served as negative and solvent controls. Body weight, clinical symptoms, and skin irritation were recorded. Gross lesions and the dorsal skin were fixed. The skin tissue from the application site was the only tissue that was examined histologically. Body weight was increased in all dosed groups; the survival time was comparable to that of the control groups. Treatment-related scale and eschar formation indicative of skin irritation were found in all 2-EHA groups beginning after the first few weeks of treatment. The subcutis was thickened and sometimes pigmented. The cutis showed hyperkeratosis, hyperplasia and scabbing in the 86.5 % and 21 % dose groups and with smaller incidence in the 43 % and 2.5 % groups. The observed skin changes were reversible in the 2.5 % group after the 11th week of treatment and in the 43 % group of the stop test immediately after treatment was stopped. Only in the 86.5 % and 21 % test groups papillomas of the skin were found and in a large percentage of animals cornified squamous cell carcinomas, melanocarcinomas and fibrosarcomas were identified without any dose dependency. No skin tumours were found in the control groups, in the groups treated with 2.5 % 2-EHA for lifetime or in the group treated with 43 % 2-EHA for about 6 months and observed for lifetime. Hepatic tumours were found in more than half of the mice of each dose group and control groups without any relation to treatment. Histologic examination of other organs was less extensive. The authors concluded that irritative skin lesions were precursors of the neoplasia. From this dermal lifetime study a LOAEL for local nonneoplastic effects on the skin was 25 µl 2-EHA solution at a concentration of 2.5 % 2-EHA in acetone was derived (24.8 mg/kg bw/day). 

 

In order to confirm the preliminary findings of the above cited study a further study with the same strain, sex and 2-EHA-concentration was done, two additional dose groups were tested (Wenzel-Hartung, 1989; Brune and Deutsch-Wenzel, 1986). In this carcinogenicity study 25µl of 2-EHA (86.5%, 21%, or 2.5% solution in acetone, approximately 1,081, 262, 31 mg/kg bw/day) was applied 3 times/week to the clipped dorsal skin of male C3H/HeJ mice (80 per group) over their lifetime. Another group was treated with a 43% 2-EHA solution for 24 weeks and thereafter observed for lifetime (stop-test). An untreated group and acetone group served as controls. Body weight, clinical symptoms, and skin irritation were recorded. Gross lesions and the dorsal skin were fixed. The skin tissue from the application site was the only tissue that was examined histologically. Body weight was increased in all dosed groups; the survival time was comparable to that of the control groups. Treatment-related scale and eschar formation indicative of skin irritation were found in all 2-EHA groups beginning after the first few weeks of treatment. The subcutis was thickened and sometimes pigmented. The cutis showed hyperkeratosis, hyperplasia and scabbing in the 86.5% and 21% dose groups and with smaller incidence in the 43% and 2.5% groups. The observed skin changes were reversible in the 2.5% group after the 11th week of treatment and in the 43% group of the stop test immediately after treatment was stopped. Only in the 86.5% and 21% test groups papillomas of the skin were found and in a large percentage of animals cornified squamous cell carcinomas, melanocarcinomas and fibrosarcomas were identified without any dose dependency. No skin tumours were found in the control groups, in the groups treated with 2.5% 2-EHA for lifetime or in the group treated with 43% 2-EHA for about 6 months and observed for lifetime. Hepatic tumours were found in more than half of the mice of each dose group and control groups without any relation to treatment. Histologic examination of other organs was less extensive. The authors concluded that irritative skin lesions were precursors of the neoplasia. From this dermal lifetime study a LOAEL for local nonneoplastic effects on the skin was 25 µl 2-EHA solution at a concentration of 2.5% 2-EHA in acetone was derived (31 mg/kg bw/day). Incidences for skin tumours in animals were as follows: 

 

 

 

 Table 5.8.3.1.Incidences for skin tumours in animals  

 

   

 

Number of animals with skin tumours	2-EHA-dose
Tumours	86.5%	43%*	21%	2.5%	Acetone control	Untreated control
Papilloma	8	-	4	-	-	-
Papilloma with strong cornification	2	-	1	-	-	-
Cutaneous horn	2	-	1	-	-	-
Haemangioma	1	-		-	-	-
Basal cell carcinoma		-	1	-	-	-
Corinified squamous-cell carcinoma	16	-	20	-	-	-
Malignant melanoma	9	-	7	-	-	-
Fibrosarcoma	-	-	5	-	-	-

 

 * Stop-test  

 

 (Note from the registrant:As mentioned above, major methodological deficiencies make the studies with the C3H/HeJ mouse strain not reliable. This will be discussed further under the “Detailed information mode of action/human relevance framework” section below.) 

  

 2-EHA was also tested for carcinogenicity in male NMRI mice exposed dermally 3 times/week to 25 µl of 21.5%, 43%, or 85% 2-EHA (w/w) diluted in acetone (approximately 269, 538 and 1,063 mg/kg bw/day) (BASF AG, 1992). 39-40 of 80 males tested were treated with 2-EHA alone, acetone (solvent control) or 0.015% (w/w) of benzo(a)pyrene in acetone (positive control, results were not reported here) for up to 24 months (BASF, 1992). Two animals of each group were killed at week 13 to examine the skin lesions. Nearly half of the animals (30-39 males) of each dose and control groups were treated as above for 7 months, after a treatment-free period of two months animals were treated with a promoter, 2-O-tetradecanoylphorbol-13-acetate (TPA), for 20 weeks. Thereafter there was no further treatment until the end of the study. Histopathology data were reported on the treated skin of all test animals and on the untreated skin area of 5-10 animals per group. Other organs/tissues were not included in histopathology examinations. 

 

 Whereas premature deaths were seen in the benzo(a)pyrene control group no treatment-related effect on mortality was observed in the 2-EHA groups. Neither 2-EHA nor the promoter TPA caused clinical signs besides the skin effects. Treatment-related lesions of the treated skin region were observed after 13 weeks and after 24 months in all 2-EHA groups. Some findings as hyperkeratosis, hyperplasia, crust formation and ulceration increased in severity or incidence related to the doses of 2-EHA, others (lymphocyte/macrophage infiltration, dermal fibrosis) were observed without any relation to the treatment groups. Reddening and thickening of the skin and similar microscopic skin lesions were reported during the promoter-phase. The authors concluded an irritative effect of the promoter TPA itself. 

 6/41 animals of the acetone group showed mild clinical symptoms (hyperkeratosis, hyperplasia, ulceration or lymphocytic/macrophage infiltrations). 

 

 (Note from the registrant: Macroscopic and histopathological interim evaluation of the skin of 2 mice from each dose group after 3 months showed scaling at the high dose, skin lesions at the low and high dose, as well as erythema and skin thickening with plication in all groups. Microscopic evidence of crust formation and ulceration were also reported at 21%; lymphocyte in¿ltration, hyperkeratosis, and hyperplasia were noted in all groups (Murphy et al, 2018). Based on these limited data, it appears that 21% likely exceeds an MTD in the NMRI mouse.) 

 

 None of animals treated with 2-EHA for up to 24 months showed a neoplastic lesion of the skin. 2-EHA and TPA promotion caused a squamous cell papilloma in one animal in each dose groups (of 30, 39, and 36 animals). One squamous cell papilloma was found in untreated skin of one male out of 41 of the acetone group. 

 

The authors concluded that these tumours were related to the irritative effects of TPA, not to the treatment with 2-EHA.The LOAEL for nonneoplastic toxic effects of 2-EHA on the skin was 25 µl 2-EHA solution at a concentration of 21.5% 2-EHA in acetone (269 mg/kg bw/day). 

 

 The findings did not indicate a carcinogenic potential of 2-EHA on the skin of male NMRI mice. 

 

 Information on cleavage products 

 

Cancer data from one oral rat study and two dermal mice studies (without conformance to requirements of the actual carcinogenicity testing protocols) using acrylic acid as test substance were considered. 

    

 In a valid carcinogenicity study (BASF AG, 1989; Hellwig et al., 1993) Wistar rats were administered to doses of 120, 400 or 1,200 ppm (mean substance uptake 9, 31, or 88 mg/kg bw/day) acrylic acid (99%, stabilised with 200 ppm hydroquinone monomethylether) in the drinking water for 26 months (males) or 28 months (females). Except a slightly reduced water consumption of high dose males and females no treatment-related clinical, hematological or histopathological changes were detected in comparison with the controls. The incidence and organ distribution of tumours found in the groups treated with acrylic acid did not differ from those of the controls. 

   

 In a dermal carcinogenicity study no tumour of the skin or subcutis were induced in treated mice or in the vehicle controls. (Intercompany Acrylate Study Group, 1982). A group of 40 C3H/HeJ male mice received 25 µl applications of acrylic acid as 1.0% (v/v) dilutions in acetone. A negative control group received acetone only. The substances were applied to the skin of the back three times weekly for the lifetime of the animals. Histologic examination was performed on the dorsal skin of all treated mice and on gross lesions. The mortality rate was not affected by treatment (mean survival time in the acrylic acid group 515 days, in the acetone group 484 days). No signs of skin irritation were observed. One male of the acrylic acid group had an epidermal hyperplasia. 

     

In another dermal carcinogenicity study 25 or 100 µl of 1% (v/v) acrylic acid in acetone was administered to two strains of mice (C3H/HeN Hsd BR, Hsd:(ICR)BR) during 21 months (3 times/week). Histopathology was done on the skin, some internal organs and every unusual gross lesion. No treatment-related signs of skin irritation, toxicity, clinical signs or skin tumours were observed. There was no treatment-related effect on body weight gain or mortality rate. 7/50 female C3H-mice of the 100 µl acrylic acid treated group revealed a significant increased frequency of lymphosarcoma compared to the acetone control group (BAMM, 1990, 1991; TSCATS, 1990, 1992a, 1992b) but lymphosarcomas are commonly seen in most strains of mice which are 18-24 months of age (Frith and Wiley, 1981) and their relation to the treatment was considered to be uncertain. 

 

 2-Ethylhexanol is known as peroxisome proliferator in animals, however this mechanism of tumour growth is considered not to be significant for humans. Results from long-term studies in rats and mice (EPA, 1992a, b) did not indicate that 2-ethylhexanol is carcinogenic in animals. Arneson et al (1995) reported that the US-National Toxicology Program nominated 2-ethylhexanol for further cancer studies. 

 

 Detailed information on mode of action / Human relevance framework: 

 

 In an expert review (Elmets and Yusuf, 2020) of the carcinogenicity studies performed on 2 -EHA and of the underlying mechanism was evaluated and it was concluded: 

 

 1) Very high doses/concentrations of 2-EHA were employed substantially greater than that to which humans are exposed. In addition, this monomer is only handled in workplaces under controlled conditions that minimize or eliminate exposure. 

 

 2) At all of the doses used, topical application of 2-EHA caused crusting, inflammation and ulcerations of the skin. 

 

 3) C3H/HeJ mice are an imperfect model for cancer assessments because they have deviant innate and adaptive immunity due to an inactivating mutation in the gene that encodes the TLR4 gene. 

 

 4) Skin lesions regressed after application of 2-EHA (43%) was discontinued. No tumors could be seen in the low dose (2.5%) group when skin irritation occurred only during the initial phases of the experiment. These observations provide strong evidence that chronic inflammation and immune dysregulation, rather than genotoxic injury was responsible for tumors that occurred (Wenzel-Hartung et al., 1989). 

 

 5) The MMTV status of the C3H/HeJ mice was not stated; if these mice carried the virus, it may have influenced the results of the skin carcinogenesis studies. 

 

 The types of tumors that developed in C3H/HeJ mice are noteworthy. Premalignant papillomas, squamous cell carcinomas, fibrosarcomas, basal cell carcinomas and melanomas were reported. The pathogeneses of these neoplasms are all different, with distinct mutations in each type. This provides additional support for the concept that the effect of 2-EHA in that strain was caused by a deviant immune response to tumor antigens caused by the TLR4 mutation. The fact that melanomas arose in 2-EHA treated mice is surprising since there were no good models of melanomagenesis until recently (Nasti et al., 2016). Thus, it is questionable as to whether they were actually tumors; it is likely that they were melanophages that had phagocytosed melanin in the dermis. There had also been no good murine models of basal cell carcinoma at the time that the study was conducted. Current methods that are used to definitively identify melanomas include immunohistochemical and/or mRNA evaluation of lesions for tyrosinase, S100, and MelanA as well as examination for compatible genetic mutations (Nasti et al., 2016).” 

 

   

  In addition, Murphy et al., (2018) highlighted that the dose selection for the DePass, (1985) screening study in C3H/HeJ mice was based on a 2-week study and tissue examination was limited to a gross evaluation. According to contemporary criteria (OECD, 2012; USEPA, 1988; USEPA, 1998) this is insufficient for proper maximum tolerated dose (MTD) definition. A subchronic dermal study was later conducted to assess the irritant properties of 2-EHA solutions in C3H/HeJ mice as a range under for the chronic studies. In the C3H/HeJ chronic study (Brune and Deutsch-Wenzel, 1986; Wenzel-Hartung et al., 1989), 12 weeks of treatment produced eschar in 67 (84%), 49 (61%) and 52 (65%) of mice administered 86.5%, 43% and 21% 2-EHA, respectively, clearly exceeding the MTD and prompting the addition of a 2.5% group. Therefore, MTD for dermal application was clearly exceeded at dose levels >21% in C3H/HeJ mice.   

   

 Using current guidance for setting the MTD, the 2.5% dose in the C3H/HeJ mouse met the criteria for the MTD, and no skin tumors developed at this dose. The dose ranges in the C3H/HeJ studies exceeded the MTD for skin integrity based on interim and terminal evaluations of the application site skin condition. Nonetheless, even studies with design ¿aws can provide useful information related to hazard (Rhomberg et al., 2007), however tumor response information in excess of an MTD should not be considered in a human hazard or risk assessment paradigm. For the purposes of an appropriate hazard assessment, 2-EHA did not cause or initiate dermal carcinogenesis in an immune competent (NMRI) mouse model (BASF AG, 1992), and, even in an immune compromised (C3H/HeJ) mouse model, did not induce skin tumors at doses which did not exceed the MTD. 

 

    

Methodological deficiencies also exist in the DePass et al., 1985 

 

 Additional references: 

 

 Elmets andYusuf (2020) Biomed Hub 2020;5:508295

 

 Murphy, S., Ellis-Hutchings, R., Finch, L., Welz, S., and Wiench, K. (2018). Critical evaluation of 2-ethylhexyl acrylate dermal carcinogenicity studies using contemporary criteria. Toxicology Letters, 294: 205-211.  

 

 Nasti, T.H., Cochran, J.B., Tsuruta, Y., Yusuf, N., McKay, K.M., Athar, M., Timares, L., and Elmets, C.A. (2016). A murine model for the development of melanocytic nevi and their progression to melanoma. Mol Carcinog 55, 646-658. 

 

 OECD, 2012. Guidance document 116 on the conduct and design of chronic toxicity and carcinogenicity studies. Supporting Test Guidelines 451, 452 and 453, 2nd edition. 13 April 2012.  

 

 Rhomberg LR, Baetcke K, Blancato J et al. (2007)Issues in the Design and Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: Approaches to Dose Selection. Critical Reviews in Toxicology, 37(9):729-837. 

 

 USEPA, 1988. Summary of the Second EPA Workshop on Carcinogenesis Bioassay via the Dermal Route, May 18-19, 1988 Research Triangle Park NC. EPA560/6-89-003. 

 

 USEPA, 1998. Health E¿ects Test Guidelines Carcinogenicity OPPTS 870.4200. EPA 712–C–98–211.