The legacy of the F344 rat as a cancer bioassay model (a retrospective summary of three common F344 rat neoplasms)

Figures & data

Figure 1. Comparison in male rats of distribution of background incidence for high incidence MNCL and LCTs versus Liver tumor incidence in (A) feeding studies and (B) Chamber studies. MNCLs have both a high incidence and wide distribution in comparison to the other tumor types which makes it difficult to determine if differences between treatment and control are treatment related or due to random chance. The difference in background incidence levels for LCTs in feeding studies (A) and chamber studies (B) maybe explains why five out of the seven NTP studies identified as having increased LCT incidence were inhalation studies. Possible biological reasons for the differences in background incidence are discussed in the text.

Figure 2. Highly variable and unpredictable nature of the MNCL background tumor incidence. Background incidence in Haseman at al., 1998 was separated by sex and study type. As can be seen above there are relatively large differences in background incidence for MNCL between sexes as well as between exposure methods (A). Contrast this with the effect sex and exposure type has on liver tumor incidence (B). Other factors that affect background incidence of MNCL are discussed in the text.

Table 1. Variables, factors and events influencing the incidence of MNCL in F344 rats.

Variable/factor/event	Effect on MNCL incidence	References
Gender	Incidence higher in male than in female controls	Haseman et al. 1985Haseman 1983Haseman et al. 1998Rao & Haseman 1993
Diet – Commercial rodent chow versus NIH-07	72% increased incidence in males fed NIH-07 diet	Rao & Haseman 1993
Diet – NIH-07 versus NTP-2000	Slight decreased incidence in males and females in NTP 2000 diet feeding studies but not in inhalation studies	Haseman et al. 2003
Diet restriction	Decreased incidence. Progression is retarded but rats live longer and eventually develop MNCL	Hursting et al. 1993Imai et al. 1990Shimokawa et al. 1993Thurman et al. 1994Turturro et al. 1994Lipman et al. 1999Stefanski et al. 1990
Corn oil gavageSafflower oil gavageTricaprylin gavage	Decreased incidence in males (up to 57% lower compared to non-gavage NIH-07 diet or water gavage); females not affected	Hursting et al. 1994Rao & Haseman 1993Haseman et al. 1985Haseman & Rao 1992NTP TR 426
Body weight at 14 weeks	Positive correlation with MNCL as adult	Turturro et al. 1994
Body weight at 12 months	Slight increased incidence in both sexes	Haseman et al. 2003
Inhalation chamber control	Increased incidence in females versus feeding study	Haseman et al. 1985, 1998, 2003
Splenectomy at 1–2 months of age	Significant decreased incidence later in life	Moloney & King 1973
Splenic toxicity	Significant decreased incidence in both sexes	Elwell et al. 1996. (Report on 13 cases with decreased incidence).
StressEpinephrine and prostaglandins		Ben-Eliyahu et al. 1999Inbar et al. 2011
Increased liver tumors	Decreased incidence	Haseman 1983Harada et al. 1990
Hypophysectomy	Decreased incidence	Ward et al. 1990Stefanski et al. 1990
Germfree versus conventional	MNCL not seen in germfree F344	Sacksteder 1976
Radiation	Decreased incidence	Hellman et al. 1982
Time trends	Increased diagnoses over time in both sexes	Rao et al. 1990Haseman & Rao 1992
Refinement of diagnostic criteria	Increased incidence in both sexes based on recognition of early stages of MNCL	Rao et al. 1990
60 Hz Magnetic field	No effect on MNCL	Morris et al. 1999Sasser et al. 1996

Table 2. Decreased frequency of MNCL in NTP studies.

NTP TR	Carcinogenic in rodents with decreased MNCL (as per Elwell et al. 1996)	Splenic toxicity	Sex
TR-351	p-Chloroaniline hydrochloride	x	M&F
TR-222	C.I. Disperse Yellow 3	x	M&F
TR-226	C.I. Solvent Yellow 14	x	M&F
TR-225	D & C Red 9	x	M&F
TR-360	N,N-Dimethylaniline	x	M&F
TR-266	Monuron	x	M&F
TR-442	p-Nitrobenzoic acid	x	M&F
TR-383	1-Amino-2,4-dibromoanthroquinone	x	M&F
TR-407	C.I. Pigment Red 3	x	M&F
TR-337	Nitrofurazone	x	M&F
TR-448	Isobutyl nitrite	x	M&F
TR-233	2-Biphenylamine hydrochloride	x	M&F
TR-205	4,4′-Oxydianiline	x	M&F
TR-271	HC Blue 1	x	M&F
TR-216	11-Aminodecanoic acid		M&F
	Not carcinogenic in rodents with decreased MNCL (as per Elwell et al. 1996)
TR-211	C.I. Acid Orange 10	x	M&F
TR-330	4-Hexylresorcinol	x	M&F
TR-411	C.I. Pigment Red 23		M&F
TR-240	Propyl gallate		M&F
TR-322	Phenylephrine hydrochoride		M&F
	Decreased MNCL (Post Elwell et al. 1996)
TR-527	Malachite green		F
TR-527	Leukomalachite green		M&F
TR-494	Anthraquinone		M&F
TR-383	1-Amino-2,4-dibromoanthraquinone		M&F
TR-493	Emodin		M&F
TR-426	Corn oil, safflower oil, tricaprylin		M

Table 3. Male and female F344 rat MNCL responses in NTP studies.

			MNCL/leukemia response
Chemicals	NTP TR	Sex	Positive		Negative
Studies evaluated prior to use of NTP’s levels of evidence of carcinogenicity
Allyl isovalerate	253	M	X
		F			X
2-Amino-5-nitrothiazole	053	M	X
		F			X
Butyl benzyl phthalate	213	M			a
		F	X
3,3′-Dimethoxybenzidine-4,4′-diisocyanate	128	M	X
		F	X
Lasiocarpine	039	M			X
		F	b
2,4,6-Trichlorophenol	155	M	X
		F			X
Chemicals	NTP TR	Sex	Cleasr	Some	Equivocal	None
Studies with clear evidence of carcinogenicity for MNCL in one or both genders
o-Nitroanisole	416	M	X
		F	X
Furan	402	M	X
		F	X
Tetrachloroethylene	311	M	X
		F		X
C.I. Direct Blue 15	397	M			X
		F	X
Tetrafluoroethylene	450	M			X
		F	X
2,2-bis(Bromomethyl)-1,3-propanediole	452	M	X
		F				X
Gallium arsenide	492	M				X
		F	X
Glycidol	374	M				X
		F	X
Mirex	313	M				X
		F	X
Studies with some evidence of carcinogenicity for MNCL in one or both genders
Dibromoacetic acid	537	M			X
		F		X
Dichlorvos	342	M		X
		F				X
Dimethyl morpholino-phosphoramidate	298	M		X
		F		X
Hydroquinone	366	M				X
		F		X
Iodinated glycerol	340	M		X
		F				X
2-Mercaptobenzothiozole	332	M		X
		F				X
Riddelliine	508	M		Xc
		F		Xc
Studies with equivocal evidence of carcinogenicity for MNCL in one or both genders
Acetaminophen	394	M				X
		F			X
Alpha methylstyrene	543	M			X
		F				X
Ampicillin trihydrate	318	M			X
		F				X
Benzophenone	533	M			X
		F			X
Chlorinated paraffins	308	M			X
		F				X
Chlorinated water	392	M				X
		F			X
Chloraminated water	392	M				X
		F			X
C.I. Acid Red 114	405	M				X
		F			X
Diallylphthalate	284	M				X
		F			X
3,3′-Dimethylbenzidine dihydrochloride	390	M				Xd
		F			Xd
Dimethyl methylphosphonate	323	M			X
		F				X
Indium phosphide	499	M			X
		F			X
4-Methylimidazole	535	M				X
		F			X
Methyl isobutyl ketone	538	M			X
		F				X
Pyridine	470	M				X
		F			X
Tris(2-chloroethyl) phosphate	391	M			X
		F			X

a = Inadequate study; b = Combined lymphoma and granulocytic leukemia; c = Study terminated at 72 weeks; d = 14-month study duration.

Table 4. NCI/NTP F344 rat studies with a positive leukemia response started and evaluated prior to introduction of NTP levels of evidence of carcinogenicity.

Two-year study highlights	Overall MNCL incidences^a	MNCL historical control data	Authors’ commentary
Allyl Isovalerate (NTP TR 253)
This corn oil gavage study started in 1979 and there was good survival and body weight gain in rats. Marginal increases in pancreatic acinar and preputial tumors were present in treated males along with an increase in MNCL. No increased incidence of tumors was seen in female rats or male B6C3F1 mice. Treated female B6C3F1 mice had increased incidence of lymphoma.	F 4/50 (8%), 6/50 (12%), 8/50 (16%)M 1/50 (2%), 4/50 (8%), 7/50 (14%)	13.2% (range 2–42%)49.6% (range 2–24%)	There was discussion during the peer review regarding how biologically relevant the leukemia response was and debate regarding which of several different historical controls should be used in the evaluation. It is noted that the incidences of MNCL are unusually low in all groups and within the laboratory historical control range.
2-Amino-5-nitrothiazole (NTP TR 053)
There was dose-related early mortality in rats in this dosed-feed study. A mixture of hematopoietic neoplasms was present in male and female rats but only considered a positive response in males. According to the TR, there was no clear evidence of carcinogenicity in female rats or in either sex of B6C3F1 mice.	All lymphomas/leukemias combined.F 2/24 (8%), 9/24 (37%), 11/24 (46%), 1/24 (4%)M 13/50 (26%), 19/50 (38%), 28/49 (57%)	No historical control data provided in TR.	The leukemia was consistent with MNCL. There was a marginally significant trend for leukemia but no significant pairwise statistical flag for MNCL. During the 1978 peer review, it was suggested that the male response might be within normal statistical variation. It is not clear why the female response was not regarded as positive.
Butyl benzyl phthalate (NTP TR 213)
This is the first of two dietary studies. This study was considered inadequate for male rats due to toxicity and early mortality. Female rats had a marginally increased incidence of MNCL as the only tumor response. There was no increased in tumor incidences in B6C3F1 mice.	F 7/49 (14%), 7/49 (14%), 18/50 (36%)M Study inadequate	19% (range 12–24%)	Peer reviewed in December 1978. Study repeated starting in 1989 (TR 458) using same or higher doses. In the repeat study there was an increase in pancreatic acinar tumors but no increase in MNCL. MNCL incidences – [F 21/50 (42%), 20/50 (40%), 21/50 (42%), 19/50 (38%); M 31/50 (62%), 28/50 (56%), 34/50 (68%), 30/50 (60%)]. We note the high background incidence of MNCL in this second study.
3,3′-Dimethoxybenzidine-4,4′-diisocyanate (NTP TR 128)
Test agent was administered by gavage and then followed by dosed-feed. Exposure was for 78 weeks plus 26 weeks without treatment. There was dose-related increased mortality in both sexes. The increased incidence of leukemia/malignant lymphoma was present along with skin and Zymbal gland tumors, and endometrial stromal polyps in treated rats. The B6C3F1 mouse study was negative for carcinogenicity.	F 1/20 (5%), 8/50 (16%), 13/48 (27%)M 0/20 (0%), 19/50 (38%), 17/50 (34%)	No historical control data provided in TR	The leukemia was morphologically characteristic of MNCL. The lymphoma diagnoses were also consistent with MNCL. The low leukemia incidence and small group size of the controls is noted. The 1978 peer review comments were supportive of the leukemia/malignant lymphoma diagnoses.
Lasiocarpine (NTP TR 039)
In this dosed-feed study, there was significant dose-related mortality in male and female rats. Liver angiosarcomas and hepatocellular tumors were treatment-related in both sexes. The leukemia was diagnosed as granulocytic leukemia. Lymphoma/leukemia was increased in low and mid-dose female rats. There was no associated mouse study.	Lymphoma/leukemia combinedF 2/24 (8%), 9/24 (37%), 11/24 (46%), 1/23 (4%)M 3/24 (12%), 3/24 (12%), 11/24 (46%), 7/24 (29%)	No historical control data provided in TR	Slides were not available to determine if the leukemia was consistent with MNCL. During the 1977 peer review by the Data Evaluation/Risk Assessment Subgroup of the Clearinghouse on Environmental Carcinogens, it was pointed out that the incidence of leukemia in treated rats was approximately the same as in female controls in a contemporary study of hexa-chlorophene. The small experimental group size was noted.
2,4,6-Trichlorophenol (NTP TR 155)
This was a dosed-feed study with good survival but lower body weight in treated rats. The leukemia in male rats was described a monocytic with mature and blast forms. Liver tumors were increased in treated B6C3F1 mice.	F 3/20 (15%), 11/50 (22%), 11/50 (22%)M 4/20 (20%), 25/50 (50%), 29/50 (58%)	No historical control data provided in TR	The monocytic leukemia was confirmed to the characteristic of MNCL. The study summary noted a large number of circulating monocytes in male and female rats.

TR: NTP Technical Report; M: Male; F: Female; MNCL: Mononuclear cell leukemia.

a = Tumor incidences arranged starting with controls and progressing through increased doses.

Table 5. NTP studies where at least one sex had MNCL classified as clear evidence of carcinogenicity.

Two-year study highlights	Overall MNCL incidences^a	MNCL historical control data	Authors’ commentary
2,2-Bis(bromomethyl)-1,3-propanediol (NTP TR 452)
This dietary administration study started in March 1989 with a high-dose male stop-study group. There were epithelial tumors in several tissues in rats and mice. There was an increase in tunica vaginalis mesotheliomas in male rats and increased incidence of MNCL. Oral cavity and esophageal tumors were present in female rats. Harderian gland and lung tumors were present in treated B6C3F1 mice.	F 15/50 (30%), 13/51 (25%), 19/53 (36%), 19/52 (37%)M 27/51 (53%), 29/53 (55%), 40/51 (78%), 34/55 (62%), 25/60 (42% – stop exposure group)	(Not provided in TR)M 48.9% ± 8.8% (range 32–62%)	The MNCL response in the top two doses of the main study was significantly different by the life table test and, therefore, MNCL was listed as part of the clear evidence of carcinogenicity in male rats. It is noted that only the mid-dose exceed the historical control range and MNCL was not increased in the stop exposure group of males.
C.I. Direct Blue 15 (NTP TR 397)
Drinking water study started in 1983 with 9 and 15-month interim sacrifices and with 22-month final sacrifice due to excessive mortality. Multisite positive carcinogen in both sexes including skin, Zymbal’s gland, liver, oral cavity, intestine, etc. MNCL was listed as clear evidence of carcinogenicity in females and as may have been related to chemical treatment in males. There was no study in mice.	F 7/50(14%), 13/35(37%), 27/65(42%), 15/50(30%)M 17/50(34%), 19/35(54%), 28/65(43%), 20/50(40%)	F 20% ± 8% (range 6–40%)M 37% ± 16% (range 10–72%)	Overall incidences within historical control ranges for both sexes. Life table statistical flags are significant. Competing risks from multiple fatal neoplasms. The wide historical control range in males makes that response uncertain. Contribution of MNCL to NTP conclusions of clear evidence in females and equivocal evidence in males is questionable.
Furan (NTP TR 402)
This corn oil gavage study started in June 1982 and included a high-dose stop-exposure. There was a high incidence of liver cholangiocarcinoma in the stop study as well as the main study. MNCL was listed as clear evidence of carcinogenicity in both sexes. There were hepatocellular and adrenal tumors in treated B6C3F1 mice.	F 8/50 (16%), 9/50 (18%), 17/50 (34%), 21/50 (42%)M 8/50 (16%), 11/50 (22%), 17/50 (34%), 25/50 (50%)	F 26.8% ± 7% (range 16–38%)M 21.3% ± 8.9% (range 4–38%)	The MNCL response exceeded the historical control range in at least one treated group in both sexes but was close to the upper historical control limit in females. There was a clear dose-response for MNCL in both sexes consistent with the NTP call of clear evidence of carcinogenicity.
Gallium arsenide (NTP TR 492)
There was good survival in this inhalation study that started in September 1993. There were no neoplastic effects in males. There were lung and adrenal tumors in addition to MNCL classified as clear evidence of carcinogenicity in females. There were no neoplastic effects in B6C3F1 mice.	F 22/50 (44%), 21/50 (42%), 18/50 (36%), 33/50 (66%)M 19/50 (38%), 28/50 (56%), 33/60 (66%), 28/50 (56%)	F 35.0% ± 5.9% (range 24–47%)M 58.0% ± 8.0% (range 42–70%)	The incidence of MNCL in females exceeded the historical control range only at the high dose and statistical significance was only p = 0.021 for this group by the poly 3 test. The control incidence of MNCL in males was unusually low and the incidence in the exposed males was within the historical control range. The call of clear evidence of carcinogenicity in females is questionable.
Glycidol (NTP TR374)
Water gavage study started in 1981. Multisite positive carcinogen in both sexes including mesothelioma, mammary gland neoplasia, glioma, oral mucosa tumors, intestine tumors, etc. Early deaths due to fatal neoplasms. MNCL in females listed as part of clear evidence of carcinogenicity. Multisite neoplasia in male and female B6C3F1 mice.	F 13/50 (26%), 14/50 (28%), 20/50 (40%)M 25/50 (50%), 33/50 (66%), 21/50 (42%)	F 20% ± 8% (range 6–40%)M 37% ± 16% (range 10–72%)	Overall incidences of MNCL within historical control ranges for both sexes. Competing risks from multiple fatal neoplasms. There was discussion of the MNCL response during the peer review. The contribution of MNCL to the NTP conclusion of clear evidence in female rats is questionable.
o-Nitroanisole (NTP TR 416)
This dietary study was started in September 1984 and included a stop-exposure group with interim sacrifices. While the stop study had urinary and intestinal epithelial neoplasia, the only tumor response in the main study was MNCL. The study was classified as clear evidence of carcinogenicity in both sexes of rats based on the combination of the main and stop studies. Liver tumors were present in the treated B6C3F1 mice.	F 14/50 (28%), 11/50 (22%), 14/50 (28%), 26/50 (52%)M 26/50 (52%), 25/50 (50%), 42/50 (84%), 34/50 (68%)	F 26.6% ± 8.8% (range 14–36%)M 48.1% ± 7/7% (range 32–62%)	This is the first NTP study where the combined results of the main and stop studies were used to classify the carcinogenic response. There was discussion during the peer review that the MNCL should be considered some evidence in males and equivocal evidence in females. Life table statistics are not highly convincing. Ultimately, the peer review supported the NTP call of clear evidence of carcinogenicity for MNCL.
Mirex (NTP TR 313)
First dietary study started in June 1977. Second dietary study at higher doses started in females 6 months later. Results combined from the two studies. Good survival. NTP reported clear evidence of carcinogenicity in males based on liver, kidney and adrenal responses and clear evidence of carcinogenicity in females based on liver and MNCL. There was no corresponding B6C3F1 mouse study.	F 14/104(13%), 8/52 (15%), 11/52 (21%), 14/52 (27%), 18/52 (35%), 27/104 (26%), 14/52 (27%)M 16/52 (31%), 17/52 (33%), 15/52 (29%), 22/52 (42%), 21/52 (40%), 10/53 (19%)	F 19% ± 7% (range 6–38%)M 29% ± 12% (range 10–60%)	MNCL response was weak in females without an early response and this was noted during the peer review. In the first study, the female MNCL was considered lethal while in the second study the female MNCL was considered incidental. Statistical flags are low and not convincing.
Tetrachloroethylene (NTP TR311)
Inhalation study started in 1981. Reduced survival in males exposed to highest concentration may have been due to MNCL. NTP reported clear evidence of carcinogenicity in male rats based on MNCL and renal tubular neoplasms and some evidence of carcinogenicity in female rats based on MNCL. B6C3F1 mice had treatment-related increased hepatocellular neoplasia.	F 18/50(36%), 30/50(60%), 29/50(58%)M 28/50(56%), 37/50(74%), 37/50(74%)	F 29% ± 6% (range 22–35%)(Laboratory)F 19% ± 7% (range 6–38%)(NTP)M 47% ± 15% (range 32–68%)(Laboratory)M 29% ± 12% (range 10–60%)(NTP)	The frequency distribution of MNCL stages 1, 2 and 3 was similar and not statistically significant among the controls and exposed rats. Comments during the NTP peer review regarding concerns about the high laboratory control rates of MNCL in this study were made suggesting conclusions regarding MNCL are questionable for both sexes.
Tetrafluoroethylene (NTP TR 450)
This inhalation study, started in June 1988, resulted in liver and kidney neoplasms in both sexes along with MNCL as clear evidence of carcinogenicity in females and equivocal evidence regarding MNCL in males. There was reduced body weight gain and increased early mortality for both sexes at the high concentration. Both sexes of treated B6C3F1 mice had liver neoplasms.	F 16/50 (32%), 31/50 (62%), 23/50 (46%), 36/50 (72%)M 34/50 (68%), 43/50 (86%), 38/50 (76%), 31/50 (62%)	F 42.0% ± 7.2% (range 30–54%)(Laboratory)40.1% ± 7.2% (range 30–54%) (NTP)56.0% ± 8.7% (range 38–66%) (Laboratory)54.4% ± 8.7% (range 34–66%) (NTP)	The control incidence of MNCL in males slightly exceeded the laboratory historical control. There was a clear increased incidence of MNCL at the low- and mid-concentrations in both sexes. The male response was considered uncertain due to the high control incidence. The female MNCL response is consistent with clear evidence of carcinogenicity.

TR: NTP Technical Report; M: Male; F: Female; MNCL: Mononuclear cell leukemia.

a = Tumor incidences arranged starting with controls and progressing through increased doses.

Table 6. NTP studies where at least one sex had MNCL classified as some evidence of carcinogenicity.

Two-year study highlights	Overall MNCL incidences^a	MNCL historical control data	Authors’ commentary
Dibromoacetic acid (NTP TR 537)
This drinking water study was started in March 2000 with good survival in treated groups. There was some evidence of carcinogenicity based on tunica vaginalis mesotheliomas in males with equivocal evidence for MNCL in males. MNCL was the only tumor response in females and was called some evidence of carcinogenicity. Liver and lung tumors were present in treated B6C3F1 mice.	F 11/50 (22%), 13/50 (26%), 16/50 (32%), 22/50 (44%)M 17/50 (34%), 31/50 (32%), 24/50 (48%), 13/50 (26%)	F 23.5% ± 4.4% (range 20–30%)M 31.6% ± 3.3% (range 26–34%)	There was significant debate regarding the MNCL call of some evidence of carcinogenicity for females during the formal public peer review and that discussion continued on the following day. It was noted that the historical control range was possibly artificially tight being based on only four studies. NTP downgraded the evidence of carcinogenicity from some to equivocal in males based on the peer review discussion.
Dichlorvos (NTP TR 342)
There was good survival in this corn oil gavage study started in January 1981 with MNCL in males and pancreatic acinar tumors in male and female rats. Forestomach tumors were present in male and female treated B6C3F1 mice.	F 17/50 (34%), 21/50 (42%), 23/50 (46%)M 11/50 (22%), 20/50 (40%), 21/50 (42%)	F (Not provided in TR)M 9% ± 7% (range 2–18%) (Laboratory)15% ± 9% (range 2–44%) (NTP)	There was discussion during the formal public peer review pointing out the higher than usual concurrent control incidence of MNCL. The MNCL response was considered contributory to the call of some evidence of carcinogenicity.
Dimethyl morpholinophosphoramidate (NTP TR 298)
In this corn oil gavage study started in April 1980, there was reduced survival in high-dose rats. The only tumor response attributed to treatment was some evidence of carcinogenicity for MNCL in both male and female rats. No treatment-associated tumors were present in B6C3F1 mice.	F 9/50 (18%), 13/5 (26%), 12/49 (24%), 18/50 (36%)M 14/50 (28%), 21/50 (42%), 19/50 (38%), 25/50 (50%)	F 29.3% ± 13.0% (range 16–42%) (Laboratory)16.1% ± 8.9% (range 2–42%) (NTP)M 17.3% ± 11.7% (range 4–26%) (Laboratory)12.2% ±7.6% (range 2–26%) (NTP)	There was increased incidence of MNCL in the high-dose for both sexes and the severity of the MNCL was greater in treated than in control rats. On the other hand, the MNCL incidence in male controls exceeded the historical control while that for the female controls was lower than the mean historical control. There was some concern voiced regarding the MNCL call during the formal public peer review.
Hydroquinone (NTP TR 366)
There was good survival in this water gavage study started in August 1982. Renal tubular tumors in the male rats and MNCL in females were considered some evidence of carcinogenicity. Liver tumors were present in female treated B6C3F1 mice.	F 9/55 (16%), 15/55 (27%), 22/55 (40%)M 28/55 (51%), 26/55 (47%), 31/55 (56%)	F 25% ± 15% (range 8–46%) (NTP)M (Not provided in TR)	It is noted that the control incidence of MNCL is lower than the historical control incidence. Some concern regarding the MNCL call was raised during the formal public peer review period due to the high and variable control incidence of MNCL. Most of the discussion, however, was focused on the male renal tumor response.
Iodinated glycerol (NTP TR 340)
This water gavage study was started in April 1981 and there was reduced survival in high-dose male rats plus some evidence of carcinogenicity for MNCL and thyroid follicular carcinomas. There were no treatment-related tumors in female rats. Female B6C3F1 mice had some evidence of carcinogenicity.	F 15/50 (30%), 14/50 (28%), 14/50 (28%)M 14/50 (28%), 29/50 (58%) 24/50 (48%)	F (Not provided in TR)M 39% ± 16% (range 14–60%)	The call of some evidence of carcinogenicity was based largely on the MNCL response in the male rats since the incidences of the thyroid tumors & nasal adenomas were marginal. There was some discussion regarding the MNCL call raised during the formal public review of the study. It is noted that the MNCL incidences for males fall within the historical control range.
2-Mercaptobenzothiazole (NTP TR 332)
There was reduced survival in treated male and high-dose females in this corn oil gavage study started in July 1981. The some evidence of carcinogenicity call in males was based on MNCL as well as preputial gland, pancreatic ascinar and adrenal pheochromocytomas. Females also had pheochromocytomas (some evidence). There was an equivocal liver tumor response in female B6C3F1 mice.	F 6/50 (12%), 14/50 (28%), 9/50 (8%)M 7/50 (14%), 16/50 (32%), 3/50 (6%)	F 19% ± 9% (range 4–42%) (NTP)M 14% ± 8%(range 2–28%) (NTP)	The MNCL call of some evidence of carcinogenicity was discussed during the formal public peer review and there were two opinions expressed that the levels of evidence for carcinogenicity in males should be equivocal. The MNCL response was present only at the low dose with poor survival at the high dose.
Riddelliine (NTP TR 508)
Riddelliine, a gavage study starting in 1996, had an unbalanced design with only a control and high-dose group for male rats. Both genders of rats are listed as clear evidence of carcinogenicity for MNCL. The male rat study was terminated at 72 weeks due to a high incidence (clear evidence) of hepatic hemangiosarcomas. Male B6C3F1 mice had hepatic hemangiosarcomas while females had lung tumors.	F 12/50(24%), 8/50 (16%), 13/50 (26%), 18/50 (36%), 18/50 (35%), 14/50 (28%)M 2/50 (4%), 9/50 (18%)	F 29.1% ± 8.4% (range 16–42%)M Not applicable. Early study termination.	The overall incidences of MNCL were within the historical control range. We have included riddelliine in our listing of some evidence based on comments made during the formal public peer review that the female MNCL response by itself should be classified as some evidence of carcinogenicity. In light of the early termination and high incidence of hepatic hemangiosarcomas in treated males, attributing any levels of evidence for MNCL carcinogenicity is questionable.

TR: NTP Technical Report; M: Male; F: Female; MNCL: Mononuclear cell leukemia.

a = Tumor incidences arranged starting with controls and progressing through increased doses.

Table 7. Occurrence of MNCL in NTP studies with at least one sex classified as equivocal evidence of carcinogenicity.

Two-year study highlights	Overall MNCL incidences^a	MNCL historical control data	Authors’ commentary
Acetaminophen (NTP TR 394)
Survival was similar among all groups in an acetaminophen 2-year study started in December 1982 at dietary concentrations of 0, 600, 3000 and 6000 ppm. The incidence of MNCL was increased in female acetaminophen groups. There was no evidence of carcinogenicity in male rats or in B6C3F1 male and females.	F 9/50 (18%), 17/50 (34%), 15/50 (30%), 24/50 (48%)M 27/50 (54%), 26/50 (52%), 24/50 (48%), 20/50 (40%)	F 16.5% ± 7/9% (range 6–28%) (Lab)F 20.8% ± 8.1% (range 6–40%) (NTP)M Not provided in TR	High dose MNCL response was considered equivocal evidence of carcinogenicity in females during NTP formal public peer review due to uncertainty that increased incidence was due to acetaminophen exposure. Lack of leukemic response in other rat studies including a F344 rat study with acetaminophen exposures up to 13,000 ppm is noted in NTP Technical Report 394.
Alpha-Methylstyrene (NTP TR 543)
This inhalation study was started in 2001. There were increased kidney tumors in males with equivocal evidence of carcinogenicity for MNCL. No increase in tumors was seen in females. B6C3F1 mice had increased liver tumors.	F 18/50 (36%), 21/50 (42%), 21/50 (42%), 22/50 (44%)M 26/50 (52%), 32/50 (64%), 29/50 (58%), 38/50 (76%)	F Not provided in TRM 47.1% ± 10.3% (range 32–66%)	No recorded comments on MNCL were documented during the formal public peer review where the focus was on kidney tumors. The male MNCL response is primarily a high-dose effect.
Ampicillin trihydrate (NTP TE 318)
This corn oil gavage study was started in 1980. There was good survival in both sexes and in mice. There was equivocal evidence of carcinogenicity in male rats based on pheochromocytomas and a marginal increase in MNCL. Female rats and B6C3F1 mice had no evidence of carcinogenicitiy.	F 14/50 (28%), 18/50 (36%), 13/50 (26%)M 5/5 (10%), 14/50 (28%), 13/50 (26%)	F Not provided in TRM 14% ± 8% (range 2–28%) (NTP)	There was considerable discussion regarding the MNCL during the formal publc peer review raising concern about how to classify the MNCL response. Ultimately a consensus vote supported equivocal evidence of carcinogenicity for the male rats. It is noted that the incidences of MNCL in males are within the historical control range.
Benzophenone (NTP TR 533)
In this dosed-feed study started in 1999, there was early mortality in high-dose males. There was an increase in renal tubular tumors in males. MNCL was considered equivocal evidence of carcinogenicity in both sexes. Mice had liver tumors (males) and histiocytic sarcoma (females).	F 19/50 (38%), 25/50 (50%), 30/50 (60%), 29/50 (58%)M 27/50 (54%), 41/50 82%), 39/50 (78%), 24/50 (48%)	F 24.6% ± 9.5% (range 12–38%)M 49.1% ± 11.9% (range 30–68%)	The extent of organ involvement with MNCL increased with dose in females and decreased with dose in males. It was noted during the formal public peer review that the incidences of MNCL in control and treated rats was unusually high.
Chlorinated paraffins (NTP TR 308)
This corn oil gavage was started in 1980. There was decreased body weight and survival in male and female rats. Liver, thyroid and kidney tumor responses were present in both sexes of rats and B6C3F1 mice.	F 11/50 (22%), 22/50 (44%), 16/50 (32%)M 7/50 (14%), 12/50 (24%), 14/50 (28%)	F 12% ± 6% (range 4–20%) (Lab)18% ± 9% (range 4–42%) (NTP)M 6% ± 6% (range 2–18%) (Lab)14% ± 8% (range 2–28%) (NTP)	The study was classified as clear evidence of carcinogenicity while the MNCL response was equivocal evidence of carcinogenicity with indication that it may have been related to treatment. It was recommended that the fact that the maximum tolerated dose was exceeded should be mentioned in the technical report abstract.
Chlorinated water (TR 392)
This drinking water study was started in 1985 with MNCL as the only tumor response attributed to treatment, seen in female rats and considered equivocal evidence of carcinogenicity. B6C3F1 mice had no evidence of carcinogenicity.	F 8/50 (16%), 7/50 (14%), 19/51 (37%), 16/50 (32%)M 25/51 (49%), 25/51 (49%), 27/50 (54%), 29/51 (57%)	F 25% ± 6.1% (range 14–36%) (NTP Feed studies)26% ± 8.5% (range 16–33%) (NTP drinking water studies)M Not provided in TR	There was considerable discussion during the formal public peer review with suggestions to downgrade the call to no evidence of carcinogenicity. However, that motion was defeated in the final voting and equivocal evidence of carcinogenicity was listed in the technical report.
Chloraminated water (TR 392)
This drinking water was started in 1985 and run along with the chlorinated water study (same TR 392). MNCL in female rats was the only response and was called equivocal evidence of carcinogenicity.	F 8/50 (16%), 11/50 (22%), 15/50 (30%), 16/50 (32%)M 25/51 (49%), 26/50 (52%), 29/51 (57%), 30/50 (60%)	F 25% ± 6.1% (range 14–36%) (NTP Feed studies)26% ± 8.5% (range 16–33%) (NTP drinking water studies)M Not provided in TR	The study is even less convincing than the chlorinated water study with all responses falling within the historical control range. A concern is with the high and variable incidence of MNCL in historic controls and the fact that study incidences were within the historic control range. The final call was equivocal evidence of carcinogenicity for MNCL in female rats.
C.I. Acid Red 114 (NTP TR 405)
This dose water study started in 1983 had early mortality due to multiple tumor sites in both sexes. MNCL in female was listed as an uncertain finding (equivocal evidence) regarding carcinogenicity. No mouse study was done.	F 12/50 (24%), 13/35 (37%), 18/65 (28%), 5/50 (10%)M 20/50 (40%), 20/35 (57%), 37/65 (57%), 12/50 (24%)	F 25% ± 6.3% (range 14–36%)M Not provided in TR	Most of the discussion during the peer review indicated a consideration that MNCL should be listed as a tumor response. Hence, it was listed as uncertain (equivocal evidence) evidence of carcinogenicity. It is noted that the incidences fall within or are just above (37% versus 36%) the historical control range. Early mortality was due to toxicity and not MNCL.
Diallylphthalate (NTP TR 284)
There was good survival in this rat only corn oil gavage study started in 1980. No evidence of carcinogenicity in male rats.	F 15/50 (30%), 15/43 (35%), 25/49 (51%)M 13/50 (26%), 12/50 (24%), 14/50 (28%)	F 29% ± 13% (range 16–42%) (Lab)16% ± 9% (range 2–42%) (NTP)M Not provided in TR	This study was called some evidence of carcinogenicity based on MNCL but was downgraded to equivocal evidence during the formal public peer review based on variability in the control incidence and difficulty in definitively diagnosing this lesion.
3,3′-Dimethylbenzidine dihydrochloride (NTP TR 390)
This was a drinking water study started in 1983 that lasted only 14 months due to mortality. Multiple tumor sites in male and female rats were documented. There was no concurrent B6C3F1 mouse study.	F 1/60 (2%), 3/45 (7%), 6/75 (8%), 4/60 (7%)M 0/60 (0%), 1/44 (2%), 1/75 (1%), 1/60 (2%).	F No historical control data for 14-month intervalM No historical control data for 14-month interval.	The frequency of MNCL is low since it is a late occurring disease in control as well as in treated rats. However, the TR speculates that MNCL may have been related to treatment (equivocal evidence of carcinogenicity) based on its possible early occurrence in treated females.
Dimethyl methylphosphonate (NTP TR 323)
This corn oil gavage study was started in 1981. Significant weight loss and decreased survival were present at the high-dose with clear evidence of carcinogenicity based on renal tumors in male and no evidence of carcinogenicity in female rats. The male B6C3F1 mouse study was inadequate and the female mice had no evidence of carcinogenicity.	F 10/50 (20%), 9/50 (18%), 12/50 (24%)M 10/50(20%), 11/50 (22%), 17/50 (34%)	F Not provided in TRM 19% ± 9% (range 4–28%) (Lab)14% ± 8% (range 2–28%) (NTP)	The increased incidence of MNCL in males was not considered part of the clear evidence call as noted during the formal public peer review and would probably fall into the category of equivocal evidence of carcinogenicity but was not indicated in the technical report. The majority of the MNCL cases were at stage 3.
Indium Phosphide (NTP TR 499)
This inhalation study was started in 1996 producing lung and adrenal tumors in both sexes. MNCL was an uncertain finding in male and female rats. Lung and liver tumors were seen in treated B6C3F1 mice.	F 14/50 (28%), 21/50 (42%), 14/50 (28%), 24/50 (48%)M 16/50 (32%), 23/50 (46%), 29/50 (58%), 25/50 (50%)	F 29.1% ± 8.5% (range 16–42%)M 43.5% ± 9.6% (range 32–54%)	There was no discussion of MNCL during the peer review. The TR report commented that the occurrence of MNCL in both sexes suggests equivocal evidence of carcinogenicity.
4-Methylimidazole (NTP TR 535)
This dosed feed study started in 2000 and there was equivocal evidence of carcinogenicity in female rats based on MNCL as the only response in rats. B6C3F1 mice had increased lung tumors.	F 9/50 (18%), 7/50 (14%), 16/50 (32%), 20/50 (40%)M 15/50 (30%), 18/50 (36%), 22/50 (44%), 20/50 (40%)	F 23.8% ± 9.1% (range 12–38%)M 46.8% ± 13.0% (range 30–68%)	MNCL was discussed during the formal public peer review and ultimately considered equivocal evidence of carcinogenicity. The female MNCL response was one tumor outside the control range.
Methyl isobutyl ketone (NTP TR 538)
This inhalation study started in 2000 resulted in kidney tumors in males and possible kidney tumors in females. Males had equivocal evidence of MNCL. B6C3F1 mice had increased liver tumors.	F 14/50 (28%), 21/50 (42%), 12/50 (24%), 16/50 (32%)M 25/50 (50%), 26/50 (52%), 32/50 (64%), 35/50 (70%)	F Not provided in TRM 47.1% ± 10.3% (range 32–66%)	No recorded comment on MNCL during the formal public peer review.
Pyridine (NTP TR 470)
This is a dosed water study started in 1991 with some evidence of carcinogenicity in males based on kidney tumors and with MNCL as the only response in females. Liver tumors were increased in male and female B6C3F1 mice.	F 12/50 (24%), 16/50 (32%), 22/50 (44%), 23/50 (46%)M 29/50 (58%), 32/50 (64%), 26/50 (52%), 27/50 (54%)	F 30.9% ± 10.0% (range 16–44%)M Not provided in TR	Female incidences of MNCL were mostly within historical control range with the high-dose just outside that range. The control incidence in a concurrent dosed water study at the same lab was 38%.
Tris(2-chloroethyl) phosphate (NTP TR391)
Survival in high dose males and females was reduced in this 2-year corn oil gavage study. Renal tubular neoplasms were increased in both sexes. Slight increases in MNCL and thyroid follicular neoplasms (equivocal evidence) were seen in both sexes. There was an interim sacrifice at 66 weeks. Male B6C3F1 had a marginal increase in neoplasms.	F 14/50(28%), 16/50(32%), 20/50(40%)M 5/50(10%), 14/50(28%), 13/50(26%)	F 15.3 ± 10.6%% (range 0–38%) (NTP)M 14.9 ± 10.8% (range 0–44%) (NTP)	NTP concluded clear evidence of carcinogenicity based on renal neoplasms and equivocal evidence based on MNCL using a life table statistical test. In males, there was a lower than expected control incidence of MNCL. In females, the MNCL incidence was marginal and restricted to the high dose group.

TR: NTP Technical Report; M: Male; F: Female; MNCL: Mononuclear cell leukemia.

a = Tumor incidences arranged starting with controls and progressing through increased doses.

Table 8. NTP studies that had a Leydig cell tumor response.

Two-year study highlights	Overall incidences^a	Historical control data	Authors’ commentary
Cumene (NTP TR 452)
Inhalation exposure to cumene was started in June 2001. There was clear evidence of carcinogenicity for nose and kidney and equivocal evidence of carcinogenicity for LCT. Lung, spleen and thyroid tumor responses were present in male B6C3F1 mice.	36/50 (72%), 38/50 (76%), 40/50 (80%), 46/50 (92%)	Mean 76.8% ± 5.9% (range 66–84%)	The judgment that the LCT response represents equivocal evidence of carcinogenicity is reasonable in that there is a clear dose response. Furthermore, the incidence of LC hyperplasia was increased in the low- and intermediate-dose groups, and the severity was increased in the high-dose group.
Ethylbenzene (NTP TR 466)
This inhalation study was started in March 1990 and had a reduced survival in high-dose males. There was clear evidence of carcinogenicity based on kidney neoplasms. The Leydig cell tumor response was also listed as clear evidence. Male B6C3F1 mice had an increase in lung tumors.	36/50 (72%), 33/50 (66%), 40/50 (80%), 44/50 (88%)	Mean 68.7% ± 8.7% (range 54–83%)	An increase incidence of unilateral and bilateral a Leydig cell tumors was slightly above the historical control in the highest concentration. There was no treatment-related increase in Leydig cell hyperplasia. At best, the Leydig cell tumor response should more appropriately be consistent with equivocal evidence of carcinogenicity
Isoprene (NTP TR 486)
This inhalation study was started in June 1993. Clear evidence of carcinogenicity was identified for mammary and kidney tumors in addition to Leydig cell tumors. There was no accompanying mouse carcinogenicity study.	33/50 (66%), 37/50 (74%), 44/50 (88%), 48/50 (96%)	Mean 69.4% ± 9.7% (range 46–83%)	Although the concurrent control is unusually low, the mid- and high-dose responses are robust. Leydig cell proliferation was present in a previously published 26-week study supporting the clear evidence of carcinogenicity call for the Leydig cell tumor response.
Kava Kava (NTP TR 571)
Kava kava extract was administered by corn oil gavage in a study that started in August 2004. There were no tumor responses in male, aside from the equivocal evidence of carcinogenicity for LCTs. Male B6C3F1 mice had an increase in liver hepatoblastomas.	37/49 (76%), 44/50 (88%), 49/50 (98%), 46/50 (92%)	Mean 88.4% ± 8.6% (range 76–94%)	The equivocal evidence of carcinogenicity for LCTs in this study was associated with a reduced latency and a dose-related decrease in Leydig cell hyperplasia. These observations suggest early conversion of hyperplasia to adenoma. It is noted that the control incidence is the lowest recorded for corn oil gavage studies.
Leucomalachite Green (NTP TR 527)
The dietary study of leucomalachite green was started in November 1998. Treatment in males was associated with equivocal evidence of carcinogenicity for thyroid and Leydig cell tumors with treatment-related decreases in pituitary adenomas and MNCL. There was no accompanying mouse study.	37/48 (77%), 42/47 (89%), 43/48 (90%), 45/47 (96%)	Mean 85.7% (range 69–90%)	The incidence of bilateral interstitial cell tumors was increased in treated rats removed from study prior to the terminal sacrifice. The call of equivocal evidence of carcinogenicity for ICT is reasonable.
Tetrafluoroethylene (NTP TR 450)
This inhalation study, started in June 1988, resulted in clear evidence of liver and kidney carcinogenesis and equivocal evidence of carcinogenesis for MNCL in males. There was reduced body weight and early mortality at the highest concentration. Bothe sexes of treated B6C3F1 mice had liver neoplasms.	39/50 (78%), 40/50 (80%), 48/50 (96%), 47/50 (94%)	Mean 68.7% ± 8.7% (range 54–83%)	The Leydig cell tumor response may have been related to treatment (equivocal evidence of carcinogenicity) but it is noted that the control incidence is within the historical control range. There was no treatment-related increase in Leydig cell hyperplasia or adenoma at a 15-month interim sacrifice.
Tetralin (NTP TR 561)
This inhalation study started in June 2003. There was some evidence of carcinogenicity based on kidney tumors and equivocal evidence of carcinogenicity for LCTs. There was no evidence of carcinogenicity in male B6C3F1 mice.	29/50 (58%), 39/50 (78%), 31/50 (62%), 41/50 (82%)	Mean 71.7% ± 8.5% (range 58–84%)	There was some discussion during the formal public peer review where it was pointed out that all of the LCT responses are within the historical control range. The judgment that the response is equivocal evidence of carcinogenicity for LCTs is based primarily on statistical evaluation. Since the concurrent control incidence of LCTs is the lowest observed in inhalation studies, the judgment of equivocal evidence of carcinogenicity for LCTs is questionable.

TR: NTP Technical Report; M: Male; F: Female; LCT: Leydig cell tumor; MNCL: Mononuclear cell leukemia.

a = Tumor incidences arranged starting with controls and progressing through increased doses.

Table 9. NTP studies that had a tunica vaginalis mesothelioma (TVM) response.

Two-year study highlights	Overall incidences^a	Historical control data	Authors’ commentary
Acrylamide (NTP TR575)
This drinking water study was started in April 2004 and yielded multiple tumor sites (pancreas, heart, thyroid) along with TVM as clear evidence of carcinogenicity. B6C3F1 male mice had Harderian gland, lung and stomach tumors.	2/48 (4%), 2/48 (4%), 1/48 (1%), 5/48 (10%), 8/48 (17%)	4.8% (range 3.3–6.4%)	Although there was no debate about the TVM response during the formal public peer review, it is noted that a TVM response was seen in two other F344 rat studies (Johnson et al. 1986; Friedman et al. 1995) but not in a Wistar rat study (Maronpot et al. 2015). The F344 rat TVM response is apparently rat strain-specific.
2,2-Bis(bromomethyl)-1,3-propanediol (NTP TR452)
This dietary study started in April 1986 had epithelial tumors in multiple tissue sites along with TVM. Tumor responses were clear evidence of carcinogenicity. B6C3F1 males had Harderian gland, skin and lung tumors.	0/51 (0%), 3/53 (6%), 8/51 (16%), 9/55 (16%), 26/60 (43%)	3% ± 2.4% (range 0–8%)	There was a clear dose related increased incidence of TVM to support the clear evidence of carcinogenicity in this study.
Bromochloroacetic acid (NTP TR549)
This drinking water study was started in July 2000. Large intestine tumors along with TVM are listed as clear evidence of carcinogenicity. Islet cell and liver tumors were considered equivocal evidence of carcinogenicity. B6C3F1 males had a liver tumor response.	1/50 (2%), 5/50 (10%), 10/50 (20%), 6/50 (12%)	3% ± 2.8% (range 0–6%)	The increased incidence of TVM in treated rats supports the clear evidence of carcinogenicity call for TVM.
1-Bromopropane (NTP TR564)
There was clear evidence of carcinogenicity for intestine and skin in this inhalation study started in July 2002. Islet cell tumors and TVM were considered to be equivocal evidence of carcinogenicity. There was no tumor response in B6C3F1 male mice.	0/50 (0%), 2/50 (4%), 2/50 (4%), 4/50 (8%)	1.4% ± 2.2% (range 0–6%)	The equivocal evidence call for TVMs is reasonable given that this tumor is rare. The high- dose incidence just barely exceeded the historical control range.
Cytembena (NTP TR207)
Cytembena was given by intraperitoneal injection in this early study that was peer review in June 1980. TVMs were present in multiple organs indicating a positive carcinogenic response. The B6C3F1 mouse study was negative for carcinogenicity.	3/50 (6%), 26/50 (52%), 26/50 (52%)	No data provided in the TR.	The increased incidence of TVM was associated with chronic peritonitis. Female rats also had peritonitis but did not have mesotheliomas.
1,2-Dibromoethane (NTP TR210)
This inhalation study peer reviewed in June 1980 had high early mortality in high-dose males. The study was positive for carcinogenicity based on nasal tumors, TVMs, and hemangiosarcomas.Male B6C3F1 mice had increased lung tumors.	0/50 (0%), 5/50 (10%), 1/50 (2%)	No data provided in the TR.	Although historical control data were apparently not available, the increased incidence of TVM in the low-dose males represented a significant increase supporting TVM as a positive carcinogenic response.
2,3-Dibromo-1-propanol (NTP TR400)
This dermal study was started in 1980 and was terminated at 55 weeks due to reduced survival. There were epithelial tumors at multiple tissue sites along with an increase in TVM as clear evidence of carcinogenicity.	0/50 (0%), 1/50 (2%), 4/50 (8%)	There were no historical control data for this 55-week duration study.	The dose-related increase in TVM, although not dramatic, should be considered in terms of the study duration and is supportive the clear evidence of carcinogenicity.
3,3′-Dimethoxybenzidine dihydrochloride (NTP TR372)
This drinking water study started in 1982. TVM were considered along with epithelial tumors in multiple sites as clear evidence of carcinogenicity. There was no corresponding mouse study.	2/60 (3%), 1/45 (2%), 7/75 (9%), 6/60 (10%)	3% (Lab); 3% ± 3% (NTP)	The dose-related increased incidence of TVM supports the clear evidence of carcinogenicity determination.
3,3′-Dimethylbenzidine dihydrochloride (NTP TR390)
This drinking water study started in 1982 had increased early mortality in all treated rats. There was clear evidence of carcinogenicity for epithelial tumors at multiple tissue sites in addition to TVM. There was no corresponding mouse study.	0/60(0%), 0/45 (0%), 3/75 (4%), 4/60 (7%)	3% (Lab); 2.9% ± 2.6% (NTP)	The modest but dose-related increased incidence of TVM supports the call of clear evidence of carcinogenicity.
Ethyl telluric (NTP TR152)
This dietary study reported in 1979 and peer reviewed at NCI had an increase in TVM as the only positive tumor response. B6C3F1 mice had Harderian gland tumors.	0/20 (0%), 2/49 (4%), 8/50 (16%)	2.9% (Lab)	Although the increased incidences in the treated rats were not statistically significant, the 16% incidence of TVM in the high-dose group clearly exceeded the laboratory historical control range indicating this study was positive for TVM carcinogenicity.
Glycidol (NTP TR374)
Glycidol was given by water gavage in 1979 and is a multisite carcinogen showing clear evidence of carcinogenicity for epithelial tumors and TVM. There was increased mortality in treated groups. B6C3F1 males had an increase of multiple epithelial tumors.	3/50(6%), 34/50 (68%), 39/50 (78%)	1% ± 2% for water gavage; 3% ± 3% for untreated rats.	Despite an unusually high control incidence, the dramatic increased incidence of TVM is supportive of clear evidence of carcinogenicity.
Methyleugenol (NTP TR491)
This gavage study used methylcellulose as the vehicle and was started in 1994. There was increased early mortality in treated groups and a 52-week stop-study. Liver tumors and neuroendocrine stomach tumors as well as an increased incidence of TVM supported clear evidence of carcinogenicity with doses up to 150 mg/kg. TVMs were also increased in the stop study (300 mg/kg). B6C3F1 mice had increased incidences of liver and neuroendocrine tumors.	1/50 (2%), 3/50 (6%), 5/50 (10%), 12/50 (24%)Stop study:1/50 (2%), 5/50 (10%)	1.7% ±2.2% (range 0–6%)	In addition to a treatment-related increased incidence of TVM, a higher dose for 52 weeks followed by only vehicle also had clear evidence of TVM carcinogenicity.
Nitrofurazone (NTP TR337)
This dietary study started in 1981 had increased mortality in the high dose. There was equivocal evidence of carcinogenicity for skin and preputial tumors as well as TVM. There were no treatment-related tumor responses in B6C3F1 males.	1/50 (2%), 7/50 (14%), 2/50 (4%)	3% ± 3% (Lab)3% ± 3% (NTP)	It is not clear why the 14% incidence of TVM was considered only as equivocal evidence of a carcinogenic response. The reduced incidence in the high-dose group reflects reduced survival.
o-Nitrotoluene (NTP TR504)
This dietary study was started in 1996 and included a 13-week stop study. There was increased early mortality in treated rats. There was clear evidence of carcinogenicity for tumors of skin, mammary gland, lung and for TVM at doses up to 2000 ppm. Male B6C3F1 mice did not have any treatment-related increase in tumors.	2/60 (3%), 20/60 (33%), 29/60 (48%), 44/60 (73%)Stop study:44/60 (73%) @ 2000 ppm54/60 (90%) @ 5000 ppm	3.7% ± 2.9%(range 0–10%)	The high incidences of TVM exclusively in male rats, including significant increases with only a 3-month exposure provide strong support for clear evidence of carcinogenicity.
Pentachlorophenol (NTP TR483)
This dietary study was started in 1992 with does up to 600 ppm and included a 52-week stop study at 1000 ppm. Some evidence of carcinogenicity for TVM was present only in the stop-study along with a questionable nasal tumor response. There was no corresponding mouse study.	Stop study: 1/50 (2%), 9/50 (18%)	3% ± 2.3% (range 0–8%)	There was considerable discussion and debate during the formal public peer review regarding the TVM response and concerns that the 1000 ppm dose in the stop-study exceeded the maximum tolerated dose and is the reason for the some evidence of carcinogenicity call.
o-Toluidine hydrochloride (NTP TR153)
This was a dermal study reported in 1979 and peer reviewed at NCI. There was early mortality in treated rats. The TVM response in the mid- and high-dose was considered positive for carcinogenicity along with subcutaneous fibromas and sarcomas in multiple organs. There was no increase in tumors in B6C3F1 mice.	0/20 (0%), 17/50 (34%), 9/49 (18%)	Not provided in the TR	The increased incidence of TVM supports the conclusion of positive carcinogenicity for o-toluidine hydrochloride.
Trimethalolpropane triacrylate (NTP TR576)
This dermal study started in 2005 and there was some early mortality in high-dose males. TVM was the only positive tumor response and was considered equivocal evidence of carcinogenicity. There was no increased tumor response in male B6C3F1 mice.	0/50 (0%), 2/50 (4%), 2/50 (4%), 5/50 (10%)	3.2% ± 3.4% (range 0–8%)	Due to the variability of control TVMs in dermal studies and the fact that the high-dose incidence was only one tumor outside the control range, this response was considered equivocal evidence of carcinogenicity.

TR: NTP Technical Report; M: Male; F: Female; TVM: Tunica vaginalis mesothelioma.

a = Tumor incidences arranged starting with controls and progressing through increased doses.

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