7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Carcinogenicity

Description of key information

A mouse skin-painting study is available.

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:

1964

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Well conducted study consistent with best practice scientific methods available at the time of conduct

Qualifier:

according to guideline

Guideline:

other: The study was performed in accordance with contemporary published methods for the assessment of dermal carcinogenicity

Version / remarks:

Holt JP, Hendricks NV, Edkardt RE, Stanton CL & Page RC (1951). A cancer-control program for high-boiling catalytically cracked oils. AMA Arch Ind Hyg Occ Med 4: 325-334.

Qualifier:

according to guideline

Guideline:

other: The study was performed in accordance with contemporary published methods for the assessment of dermal carcinogenicity

Version / remarks:

Twort CC & Twort JM (1933). Suggested methods for the standardization of the carcinogenic activity of different agents for the skin of mice. Am J Cancer 17:293-320.

Qualifier:

according to guideline

Guideline:

other: The study was performed in accordance with contemporary published methods for the assessment of dermal carcinogenicity

Version / remarks:

Spicer CC (1947). The estimation of tumour susceptibility in pure lines. Brit J Cancer 1:298-310.

Qualifier:

according to guideline

Guideline:

other: The study was performed in accordance with contemporary published methods for the assessment of dermal carcinogenicity

Version / remarks:

Berenblum I (1945). A system of grading carcinogenic potency. Cancer Res 5:561-564.

Qualifier:

according to guideline

Guideline:

other: The study was performed in accordance with contemporary published methods for the assessment of dermal carcinogenicity

Version / remarks:

Blanding FH, King WH Jr., Priestly W Jr & Rehner J Jr. (1951). Properties of high-boiling petroleum products: quantitative analysis of tumor-response data obtained from the application of refinery products to the skin of mice. AMA Arch Ind Hyg Occ Med. 4:335-345.

Principles of method if other than guideline:

The study was performed in accordance with contemporary published methods for the assessment of dermal carcinogenicity, involving chronic dermal (skin painting) exposure in mice. Methods for this study were developed in the late 1940s-1950s by a group of scientists from Standard Oil Company, Esso Standard Oil Company, Lago Oil and Transport Company, Ltd, Humble Oil and Refining Company, Imperial Oil Company, Ltd., New York University-Bellevue Medical Center, and Memorial Hospital, New York, who investigated the effects of various petroleum distillates on skin tumour production. Methods were developed in an effort to define recommended industrial hygiene practices for petroleum workers who were assumed to cleanse skin less than once daily, and who could thus potentially be dermally exposed to petroleum products for extended periods during the workday and beyond (Holt et al., 1951). Methods were based on investigatory work conducted over the previous 20 years (Twort and Twort, 1933; Spicer, 1947; Berenblum, 1945). Choice of frequency of skin painting was based on anticipated potency of the material: 1x per week, high potency; 3x per week, lower potency; 3x to 6x/week, lowest potency (Dr. A. Wesley Horton, Kettering Laboratory, College of Medicine of University of Cinncinnati, personal communication in Union Carbide Corp. study 14-60).
Groups of 40 male mice were painted with a brushful (~ 0.-0.2 g; series 197 #1 Grumbacher brush) of the substance three times a week during their lifetimes; the substance was applied to the clipped intrascapular region of the backs. Backs were clipped once a week to ensure the application surface was clear of hair. The mice were usually painted on Mondays, Wednesdays, and Fridays, in an area ~1 cm in diameter above the shoulder blades. Each painting consisted of a single brush-stroke against the direction of the hair. Mice were examined on dosing days, and the number of surviving mice bearing papillomas or cancers recorded. Notes were kept on the position of tumours, course of progression of the tumours, and necropsy results. Tumours were examined microscopically.

GLP compliance:

no

Remarks:

the study pre-dates the introduction of GLP

Specific details on test material used for the study:

The test material, as defined in this study, is named EP-221, and is composed of pure 7-OXABICYCLO(4.1.0)HEPTANE-3-CARBOXYLIC ACID, 7-OXABICYCLO(4.1.0)HEPT-3-YLMETHYL ESTER. The submission substance, ERL-4221, is composed of 82-89% 7-OXABICYCLO(4.1.0)HEPTANE-3-CARBOXYLIC ACID, 7-OXABICYCLO(4.1.0)HEPT-3-YLMETHYL ESTER.

Species:

mouse

Strain:

C3H

Details on species / strain selection:

This strain of mouse was commonly used in the skin painting studies.

Sex:

male

Details on test animals or test system and environmental conditions:

Male C3H/Anf mice were obtained from Cumberland Farms, TN, at approximately 4 weeks of age and were first dosed when approximately 3 months of age. Mice were group-housed 10/cage for the course of the study; partially depleted cages were never recombined. Mice received a standard rodent chow to eat and municipal tap water to drink.

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

Groups of 40 male mice were painted with a brushful (~ 0.1-0.2 g; series 197 #1 Grumbacher brush) of the substance three times a week during their lifetimes; the substance was applied to the clipped intrascapular region of the backs. Backs were clipped once a week to ensure the application surface was clear of hair. The mice were usually painted on Mondays, Wednesdays, and Fridays, in an area ~1 cm in diameter above the shoulder blades. Each painting consisted of a single brush-stroke against the direction of the hair.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Undiluted test material was used for the study; therefore no dose solution checks were necessary.

Duration of treatment / exposure:

Lifetime (until moribund or dead) - up to 29 months

Frequency of treatment:

Three times per week, usually Monday, Wednesday, and Friday.

Post exposure period:

None

Dose / conc.:

4 000 mg/kg bw/day (nominal)

Remarks:

The applied dose was estimated to be 4000-8000 mg/kg bw, based on 0.1-0.2 g applied to a mouse of 25 g bodyweight

No. of animals per sex per dose:

40 males

Control animals:

yes

other: A control group was treated with acetone, the vehicle used for the positive control group

Details on study design:

Methods for this study were developed in the late 1940s-1950s by a group of scientists from Standard Oil Company, Esso Standard Oil Company, Lago Oil and Transport Company, Ltd, Humble OIl and Refining Company, Imperial Oil Company, Ltd., New York University-Bellevue Medical Center, and Memorial Hospital, New York, who investigated the effects of various petroleum distillates on skin tumour production. Methods were developed in an effort to define recommended industrial hygiene practices for petroleum workers who were assumed to cleanse skin less than once daily, and who could thus potentially be dermally exposed to petroleum products for extended periods during the workday and beyond (Holt et al., 1951). Methods were based on investigatory work conducted over the previous 20 years (Twort and Twort, 1933; Spicer, 1947; Berenblum, 1945).

Positive control:

3-methylcholanthrene (3-MC)

Observations and examinations performed and frequency:

The study focussed on the potential of the test material to cause skin tumours; other observations were limited.

Mice were examined on treatment days for general health and well-being, and observed monthly for tumour incidence. Whenever a growth was first noted by handlers, its nature was verified by the lead scientist and its position noted diagramatically. Progression or regression of the growth was documented similarly. The number of surviving mice in a given dose group was documented for each observation point.

Sacrifice and pathology:

Moribund or dead mice were submitted for necropsy and the results recorded. Tumours were preserved and examined histologically for confirmation of tumour type.

Other examinations:

Data recorded in support of statistical analysis (Blanding et al., 1951):
Original number of mice per dose group
Time of observation (expressed in days) from the commencement of skin painting)
Number of deaths in each interval of observation
Number of tumour-free animals that died in each interval
Number of tumour (papilloma)-bearing animals that died in each interval
Number of cancer (carcinoma)-bearing animals that died in each interval
Total number of survivors at each time of observation
Number of tumour-free survivors at each time of observation
Number of tumour (papilloma)-bearing survivors at each time of observation
Number of cancer (carcinoma)-bearing survivors at each time of observation

Statistics:

For each time of observation, the percent tumor response was computed according to the following equation:
Percent tumour response was defined as the total number of mice with tumours/effective number of mice
The total number of mice with tumours was defined as the cumulative number of mice that died with papillomas and/or carcinomas up to the time of observation plus the current number of survivors having papillomas and/or carcinomas. That is, the numerator measures the total response for both living and dead mice, the word tumour was used without differentiation between benign and malignant forms. The denominator is computed as the original number of mice less the number that have died without any tumours up to the time of observation.
Similarly, a percent cancer response was calculated for each time of observation.
Percent tumour or percent cancer response was plotted against time logarithmically to obtain a dose-response line (probit plot). Tumour potency was then calculated as the tumour-response rate as follows:
Tumour potency = 10,000 / days to 50% tumour response (Finney, 1947). The denominator was derived from the logarithmic plot described above. An analogous calculation was done for cancer potency. Average latency period was calculated by plotting the percent response indices against time and determining the time to achieve a 50% response via a least-squares method. This was compared for the test substance vs. negative and positive controls using standard categorical analysis for the time period.

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Only one skin tumour was seen (this after 23 months of painting) and this was less than the 2 tumours seen on mice that received acetone paintings

Other effects:

not specified

Details on results:

Only one tumour was seen in the treated group (after 23 months of painting) and this was less than the 2 tumours seen on the control mice that received acetone paintings. Therefore, undiluted EP-221 was not, by this test, tumorigenic to the skin of mice.

EP-221: Number of mice alive at one year, 39; 18 months, 31; 24 months, 12
Appearance of tumour: Month 23 of painting, 15 mice still alive
Tumour index: 6.7
Cancer index: 0.0
Average latency period for tumours: >29 months
Average latency period for cancer: n/a

Acetone control:
Number of mice alive at one year, 32; 18 months, 26; 24 months, 4.
Appearance of tumours: month 23 of painting, 5 mice still alive.
Tumour index: 40.0
Cancer index: 0.0
Average latency period for tumoursl >26 months
Average latency period for cancer; n/a

Positive control methyl cholanthrene (0.2% in acetone):
Number of mice alive at one year, 2; 18 months: 0 ; 24 months, 0
Appearance of tumours: Month 3 of painting, 40 mice still alive
Tumour index: 100.0
Cancer index: 94.9
Average latency period for tumours; 5.5 months
Average latency period for cancer; 7.2 months

Relevance of carcinogenic effects / potential:

Only one tumour was seen in the treated group (after 23 months of painting) and this was less than the 2 tumours seen on control mice that received acetone paintings. Therefore, undiluted EP-221 was not, by this test, tumorigenic to the skin of mice.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 4 000 - <= 8 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Key result

Critical effects observed:

no

One mouse in the substance treatment group developed a skin tumour; the tumour was first observed from 26 months (i.e. after 23 months of treatment). Two control mice developed skin tumours; the latency period for these tumours was the same as for the treated group. All of the tumours in the treated and control groups were characterised histopathologically as papillomas. A total of 39 mice in the positive control group were observed to develop skin tumours. Tumours were observed from as early as 3 months and the vast majority (37 of 39 tumours) were characterised histopathologically as carcinomas.

Mouse skin-painting carcinogenicity study: summary of findings

Material	Concentration	Number^*alive at			Appearance of First Tumour		Total no of mice with		Max No of Months Painted	Tumour Index	Cancer Index	Average Latency (mo)
		12 mo	18 mo	24 mo	Month	No alive	Tumours	Cancer				Tumour	Cancer
EP-221	Undiluted	36	35	17	23	15	1	0	29	6.7	0.0	--	--
Acetone	Undiluted	32	26	4	23	5	2	0	26	40.0	0.0	>26	--
3MC	0.2% in acetone	2	0	0	3	40	39	37	13	100.0	94.9	5.5	7.2
*40 mice started in each group

 

Conclusions:

Only one skin tumor developed in mice exposed to ~4000-8000 mg/kg/application of EP-221 for a lifetime. The tumor index was 6.7, and the average latent period was greater than 29 months. This compares to two tumors seen with application of the negative control, acetone, and 39 seen with the positive control, methyl cholanthrene.

Executive summary:

In this study, the substance EP-221 (a purified form of the submission substance) was applied undiluted three times a week to the same position on the shorn dorsal skin of a group of 40 male C3H/Anf mice, aged 3 months. The substance was applied at a rate of 100-200 mg/mouse, calculated to be equivalent to 4000-8000 mg/kg bw. Application continued until death (or moribundity), which was up to 29 months (i.e. mice were treated for up to 26 months). A positive control group was similarly treated with 3‑methylcholanthrene (0.2% in acetone) and a negative (vehicle) control group was treated with acetone. Mice were observed daily and were assessed monthly for the development of skin tumours. Tumours were monitored for progression or regression and were assessed histopathologically as papillomas or carcinomas following the death of the animal. One mouse in the substance treatment group developed a skin tumour; the tumour was first observed from 26 months (i.e. after 23 months of treatment). Two control mice developed skin tumours; the latency period for these tumours was the same as for the treated group. All of the tumours in the treated and control groups were characterised histopathologically as papillomas. A total of 39 mice in the positive control group were observed to develop skin tumours. Tumours were observed from as early as 3 months and the vast majority (37 of 39 tumours) were characterised histopathologically as carcinomas. The results of this study therefore demonstrate an absence of skin carcinogenicity for the test material. The incidence, type and latency of skin tumours seen in this study were comparable to the control group. In contrast, administration of the known carcinogen 3-methylcholanthrene resulted in a very large proportion of treated mice with skin tumours. The study is relatively old and was not performed to current guidelines for the assessment of carcinogenicity. The study is, however, considered to be scientifically robust and was performed to a method suitable for the assessment of local (site of contact) carcinogenicity which was in widespread use at the time. The study is considered to be adequate in demonstrating a lack of carcinogenicity following repeated dermal application of the substance to mouse skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

4 000 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

The study is relatively old and was not performed to current guidelines for the assessment of carcinogenicity. The study is, however, considered to be scientifically robust and was performed to a method suitable for the assessment of local (site of contact) carcinogenicity which was in widespread use at the time. The study is considered to be adequate in demonstrating a lack of carcinogenicity following repeated dermal application of the substance to mouse skin.

System:

integumentary

Organ:

skin

Justification for classification or non-classification

No evidence of carcinogenicity was seen in the single available study. No classification is therefore proposed for cacrcinogenicity according to the CLP Regulation