Nitrosodiphenylamine

Carcinogenicity

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A bioassay of N-nitrodiphenylamine for possible carcinogenciity was conducted by administering the test chemical in feed to F344 rats and B6C3F1.

GLP compliance:

no

Species:

other: rats and mice

Strain:

other: F344 rats and B6C3F1 mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:NCI frederick cancer research center animal farm
- Age at study initiation: 4 wk-old weanlings
- Weight at study initiation: males rats = 90-105 g ; female rats = 80-95 g; male mice = 18-22 g; female mice = 17-21g
- Housing: by 4 (rats) or 5 (mice) in polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 45-55
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

Test diets containing test item were preared approximately weekly in 9- to 12-kg batches at appropriate doses.
A know weight of the chemical was first mixed with an equal weight of autoclaved with 4% fat using a mortar and pestle. The mixing was continued with second and third additions of feed, and final mixing was performed with the remaining quantity of feed for a minimum of 15 minutes in a blender. The diets were routinely stored at 7°C until used.

Duration of treatment / exposure:

Rats : 100 weeks
Mice : 101 weeks (males), 38 + 60 weeks (females)

Frequency of treatment:

daily in feed

Dose / conc.:

1 000 ppm

Remarks:

(rats)

Dose / conc.:

4 000 ppm

Remarks:

(rats)

Dose / conc.:

10 000 ppm

Remarks:

(male mice)

Dose / conc.:

20 000 ppm

Remarks:

(male mice)

Dose / conc.:

5 000 ppm

Remarks:

(female mice), then reduced to 1000 ppm from week 39

Dose / conc.:

10 000 ppm

Remarks:

(female mice), then reduced to 4000 ppm from week 39

No. of animals per sex per dose:

Rats: 50 animals/ sex/treated group. Control : 20 animals/ sex
Mice: 50 animals/ sex/treated group. Control : 20 animals/ sex

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Subchronic feeding studies were conducted to estimate the maximum tolerated doses of test item, on the basis of 2 concentrations (low and high doses) were selected for the chronic studies. Groups of 5 rats and 5 mice of each sex were fed diests containing test item at one of several doses, whilde groups of 5 control animals of each specie and sex were administered basal diet only. The length of the study in male rats was 11 weeks, while in female rats and male/female mice it was 8 weeks. Each animal was weighed twice per week.
In the first study (in male rats), no mortality was observed at 1000, 2000, 3000, 4000, 6000, 8000, 10000 ppm, with a mean weight at week 11 as 81-94% of control.
In the first study (in female rats), no mortality was observed at 4000 and 8000 ppm, with a mean weight at week 7 as 86% of control. At 16 000 ppm, 2 females died from week 5 (mean weight of 63% of control). At 24000, 32000 and 46000 ppm, all females died from week 4, 4, and 2 respectively.
In the first study (in mice), no mortality was observed at 3160, 4640, 6800, 10000 and 14700 ppm in males and females, with a mean weight at week 7 as 104-108% (males) and 88-97% (females) of control.
In the second study (in mice), no mortality was observed at 4250, 7500, 8500, 9500, 11000, 22000 ppm in males, et 22000, 32000 and 46000 in females with a mean weight at week 7 as 86-97% (males) and 86-94% (females) of control.

At the end of the subchronic studies, all animals were killed sing CO2 and necropsied. The only histopathologic lesions observed ware trace amounts of pigmentation of Kuffer's cells in hepaticx sinusoids of male mice at 46 000 ppm.
A10% depression in mean body weight was the major criterion for estimation of MTD's.

Positive control:

no

Observations and examinations performed and frequency:

All animals were oberved twice daily, and the occurrences of sick, tumor-bearing, and moriund animals were recorded. Clinical examination and palpation for masses were performed each month, and the animals were generally weight at leat once per month, except for the period of week 42 though week 64, when weights were not recorded for the rats.

Sacrifice and pathology:

Moribund animals and animals that survived to the end of the bioassay were killed using CO2 and necropsied.
The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions. The tissues were preserved in 10% buffered formalin, ambedded in paraffin, sectional, and stained with hematoxylin and eosin. the following tissues were examined microscopically: skin, lungs and bronchi, trachea, bone marrow (femur), spleen, lymph nodes (mesenteric and submandibular), thymus, heart, salivary glands, liver, pancreas, esophagus, stomach, small and large intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate, mammary gland, uterus, ovary, brain, and all tissues masses. Peripheral blood smears also were made for all animals, whenever possible.
Necropsies were also performed on all animals found dead, unless precluded in whole or in part by autholysis or cannibalization.

Statistics:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

RATS: Corneal opacity occurred at higher incidences in high-dose males (15/20) and low dose females (16/50)than in corersponding control males (0/20) and control females (1/20) and may have been related to the administration of the test chemical.

MICE: Tissue masses occurred at low incidences in both control and dosed groups of mice.

Mortality:

mortality observed, treatment-related

Description (incidence):

RATS: The results of the Tarone test for dose-related trend in mortality is not significant in male rats. In females, the results of the Tarone test is significant.
In male rats, 43/50 (86%) of the high-dose group, 44/50 (88%) of the low dose groups, and 16/20 (80%) of the control group lived to the end of the study.
In females, 35/50 (70%) of the high-dose group, 44/50 (88%) of the low dose groups, and 18/20 (90%) of the control group lived to the end of the study.
Sufficient numbers of rats of each sex were at risk for the development of late-appearing tumors.

MICE: The results of the Tarone test for dose-related trend in mortality is not significant in either sex.
In male mice, 41/50 (82%) of the high-dose group, 46/50 (92%) of the low dose groups, and 18/20 (90%) of the control group lived to the end of the study.
In females, 31/50 (62%) of the high-dose group, 42/50 (84%) of the low dose groups, and 16/20 (80%) of the control group lived to the end of the study.
Sufficient numbers of rats of each sex were at risk for the development of late-appearing tumors.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

RATS : Mean body weights of dosed male and female rats were lower than those of corresponding controls, and were dose related throughout the bioassay for the males, but only sometime following week 40 for the females. Some fluctuation in the growth curves way be due to mortality; as the size of a group diminishes, the mean body weight may be subject to greater variation.

MICE: Mean bodyweight of dosed male and female mice were lower than those of corresponding controls, especially for the females, and were dose related throughout the bioassay. Some fluctuation in the growth curves may be due to mortality; as the size of a group diminishes, the mean body weight may be subject to greter variation.

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

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see in the section "histopathological findings: neoplastic"

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see in the section "histopathological findings: neoplastic"

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

RATS: A variety of neoplasms are represented in both dosed and control groups of rats. Most of the types of tumors represented have been encountered previously in control aging F344 rats. There was a high incidence of tumors of the urinary bladder in the high-dose groups of each sex. In high-dose groups of each sex, the entire spectrum from transitional-cell hyperplasia to transitional-cell carcinoma was observed in the urinary bladder. The hyperplastic foci consisted of enlarged transitional cells and the epithelium was several (7 to 10) layers in thickness. In some of the hyperplastic foci there was a tendency for the cells in the basilar layer to compartmentalize and form circular, almost acinar-like structures. Mitotic figures were often noted in these basilar cells. As the epithelium increased the thickness, fibrous tissue strands began to appear, forming a connective tissue stroms for the proliferating epithelium. These lesions were diagnosed as transitional-cell neoplasms. The epithelium covering these fibrous stands was several (7 to 10) layers thick and mitotic figures were quite numerous in most cases. Many of the tumors were similar to papillomas, however, the thickness and activity of the epithelium was consistent with papillary transtional-cell carcinoma. Many of the tumors had less fibrous stroma, the mass consisted of solid sheets of epithelial cells, or was occasionally arranged in cords. In three cases, there was squamous metaplasia. The base of the tumor was narrow in may cases, but was also rather broad in many others. The degree of infiltration int deeper layers of the bladder wall was also variable. There appeared to be a tendency for the tumor mass to remain rather superficial until the mass was quite large in size and the tumor cells were more anaplastic and active. At this time, there was infiltration into the deeper layers; however, in only one case was there invasion through the entire wall and beyond the serosa. In none of these animals was a transitional-cell metastatic focus seen in another organ.
A second type of tumor, fibroma of the subcutis and skin, was observed at a higher frequency in the male high-dose group (10/45) than in the male low_dose group (1/46), in the male controls (1/19) or in any of the female groups. The fibromas were composed of well-differentiated, dense, well-circumscribed areas of fibrous tissue.
A variety of nonneoplastic lesions were represented among both control and dosed groups of animals. Such lesions have been encountered previously in untreated aging F344 rats and are not considered to be compound related.
CONCLUSION (rats): Based on the histopathologic examination, the high incidence of bladder tumors in both male and female high-deose groups indicates that nitrosodiphenylamine was carcinogenic for F344 rats under the conditions of this bioassay.

MICE: There were similar incidences and types of tumors in control and dosed mice, and none appeared to be related to administration of the test chemical. However, there was a high incidence of bladder lesions in the dosed mice. The lesions most frequently observed in the bladders of the mice was chronic submucosal inflammation. No such lesion was observed in any control animal. An occasional focus of lymphocytes, which are not an uncommon finding in the submucosa of the urinary bladder of normal micen were observed in the control animals in this study. In the doses animals diagnosed as having chronic submucosal inflammation of the bladder, there was an increase in the number of lymphocytes which was manifested by increased size and number of lymphocytic foci, infiltration of lymphocytes between collagen fibers, and more numerous blood vessels cuffed with lymphocytes. These lymphocytic cuffs were also much greater in thickness.
A degeneration of the collagen fibers of the submucosa was observed only in dosed mice. The degeneration was characterized by skrinking and curling of collagen bundles, and they had more hyalinized appearence. This change appeared to occur first near, or immediately beneath, the basement membrane of the epithelium and extended to varying depths in the mucosa. Deeper in the submucosa the collagen bundles were more plump, but also more hyalinized. Blood vessels themselves also had a change which was more evident in small arterioles. This change was a thickening of the media woth hyalization of the muscle fibers. In the majority of cases, this was not a severe change, but it was indeed observable. In two cases(high-dose females), this change was accompnied by acute and chronic inflammatory foci in the vessel wall and in still another case, again a high-dose female, there was fibrinoid necrosis of the vesse wall. Overall, the submucosa of the bladder seemed sowewhat thickened and in two cases was considered to be edematous. Although a few changes were observed in the epithelium of the bladder, it is somewhat surprising that more were not seen in view of the changes in the submucosa. The hyperplasia of the epithelium usually occurred as focal areas ; however, in one case it occurred as diffuse hyperplasia. Two transitional-cell carcinomas of the bladder were encountered, one of which occurred in a low-dose male, the other in a low-dose female. One transitional-cell papilloma also was seen, in a high-dose male.
A slight perivascular lymphocytic cuffing in the kidney is a normal finding in B6C3F1 mice. There were a few animals, both control and dosed, in which the degree of cuffing was considered to be greater than usual; these were diagnosed as having chronic inflammation. There was no correlation between these kidney lesions and changes in the urinary bladder.
In addition to the bladder lesions, a large number of degenerative, proliferative, and inflammatory changes were also encountered in animals of the dosed and control groups. Again no correlation could be made between incidences of lesions and administration of the test material.
CONCLUSION (MICE) : Because the incidence of bladder neoplasms encountered in the dosed mice of this study was very low, it does not appear that Nitrosodiphenylamine was carcinogenic for B6C3F1 mice under the conditions of this bioassay. The compound does, however, produce a non-suppurative inflammatory response associated with a connective tissue degeneration in the submusa of the urinary bladder in B6C3F1 mice.

Key result

Dose descriptor:

NOAEL

Remarks:

(carcinogenicity) RAT

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: assuming an intake of 20g of food per day by each 400 -g rat (values used by EFSA for older-rat), 4000 ppm corresponds to 200 mg/kg, and 1000 ppm to 50 mg/kg/day.

Dose descriptor:

NOAEL

Remarks:

(carcinogenicity) MICE

Effect level:

> 20 000 ppm

Based on:

test mat.

Sex:

male

Remarks on result:

other: not determinable beacuse of absence of tumors

Dose descriptor:

NOAEL

Remarks:

(carcinogenicity) MICE

Effect level:

> 4 000 ppm

Based on:

test mat.

Sex:

female

Remarks on result:

other: not determinable beacuse of absence of tumors

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 1: Urinary bladder lesions in rats

	Males			Females
	Control	Low dose	High dose	Control	Low dose	High dose
Transitional cell carcinoma (including papillary)	0/19	0/46	17/45 (38%) P<0.001	0/18	0/48	42/49 (86%) P< 0.001
Transitional cell carcinoma with squamous metaplasia	0/19	0/46	1/45 (2%)	0/18	0/48	2/49 (4%)
Epithelial hyperplasia	0/19	2/46 (4%) NS	6/45 (13%) NS	0/18	4/48 (8%) NS	7/49 (14%) NS

NS = p< 0.05

Table 2: Urinary bladder lesions in mice

	Males			Females
	Control	Low dose	High dose	Control	Low dose	High dose
Transitional cell carcinoma	0/18	1/49	0/46	0/18	1/47 (2%) NS	0/38
Transitional cell papilloma	0/18	1/49	1/46	0/18	0/47	0/38
Epithelial hyperplasia	0/18	2/49 (4%) NS	7/46 (15%) NS	0/18	3/47 (6%) NS	6/38 (13%) NS
Inflammation chronic submucosal	0/18	12/49 (24%) NS	31/46 (67%) P<0.001	0/18	31/47 (66%) P< 0.001	30/38 (79%) P< 0.001

NS = p< 0.05

 

 

Conclusions:

It is concluded that under the conditions of this bioassay, nitrosodiphenylamine was carcinogenic for both sexes of F344 rats, inducing transitional-cell carcinomas of the urinary bladder, but was not carcinogenic for B6C3F1 mice of either sex.

Executive summary:

A bioassay of N-nitrodiphenylamine for possible carcinogenicity was conducted by administering the test chemical in feed to F344 rats and B6C3F1.

Groups of 50 rats of each sex were administered test item at one of two doses, either 1000 or 4000 ppm, for 100 weeks. Matched controls consisted of 20 untreated rats of each sex. All surviving rats were killed at the end of administration of the test chemical.

Groups of 50 male mice were administered test item at one of 2 doses, either 10 000 ppm or 20 000 ppm for 101 weeks. Groups of 50 female mice were administered test item at one of 2 doses, initially 5000 or 10 000 ppm, for 38 weeks. Because of excessive depression in the amount of mean body weight gained in the doses groups, the doses for females were then reduced to 1 000 and 4 000 respectively, and administration at the lowest doses was continued for 60 weeks. The time-weighted average doses for the female mice were either 2.315 or 5.741 ppm. matched controls consisted of 20 untreated mice of each sex. All surviving mice were killed at the end of administration of the test chemical.

Mean body weights of doses rats and mice of each sex were lower than those corresponding controls, and were dose related throughout the bioassay, except those of female rats during the first part of the bioassay. Mortality was dose related in the female rats, butwas not affected when the test chemical was administered to the male rats or the male and female mice. Survival at the end of the bioassay was 64% or grated in the dosed and control groups or rats and mice of each sex, and sufficient numbers of animals were at risk in all groups for the development of late-appearing tumors.

Transitional-cell carcinomas of the urinary bladder occurred at incidences that were dose related (p<0.001) in both male and female rats, and in direct comparisons the incidences of these tumors in the high-dose groups of each sex were significantly higher (p<0.001) than those in the corresponding controls (males: control 0/19, low-dose 0/46, high-dose 16/45; females : controls 0/18, low-dose 0/48, high dose 40/49). The possible mechanism by which these tumors were induced, such as calculi formation in the bladder or nitrosation of amines present in feed to a carcinogenic nitrosamine, is unknown.

Fibromas of the integumentary system occurred in male rats at incidences that were dose related (p=0.003), although in direct comparisons the incidences of these tumors in the individual dosed groups were not significantly higher than those in the control group (controls 1/20 = 5% ; low dose 1/50 = 2%; high dose 10/50 = 20%). The incidence of fibromas of the integumentary system in historical-control male F344 rats at this laboratory is 6/285 (2%). These results suggest an association of the fibromas in the male rats with administration of the test chemicals.

No tumors occurred in the mice of eihter sex at incidences that were significantly higher in the dosed groups than in the corresponding control groups. The onyl changes related to coumpound administration were chronic inflammatory lesions int he urinary bladders of dosed mice.

It is concluded that under the conditions of this bioassay, nitrosodiphenylamine was carcinogenic for both sexes of F344 rats, inducing transitional-cell carcinomas of the urinary bladder, but was not carcinogenic for B6C3F1 mice of either sex.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The key study is considered to be reliable with a klimish score of 2.

System:

urinary

Organ:

bladder

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No human data is available to evaluate the carcinogenicity of nitrosodiphenylamine. Based on the NCI bioassay, there are evidences for the carcinogenicity of nitrodiphenylamine in rats after diet exposure.

In conclusion, Nitrosodiphenylamine should be classified as carcinogen Category 2 according to the Regulation EC n°1272/2008.

Target organ: bladder.

Additional information

2 year carcinogenicity study in rats and mice (NCI 1979) :

A bioassay of N-nitrodiphenylamine for possible carcinogenicity was conducted by administering the test chemical in feed to F344 rats and B6C3F1.

Groups of 50 rats of each sex were administered test item at one of two doses, either 1000 or 4000 ppm, for 100 weeks. Matched controls consisted of 20 untreated rats of each sex. All surviving rats were killed at the end of administration of the test chemical.

Groups of 50 male mice were administered test item at one of 2 doses, either 10 000 ppm or 20 000 ppm for 101 weeks. Groups of 50 female mice were administered test item at one of 2 doses, initially 5000 or 10 000 ppm, for 38 weeks. Because of excessive depression in the amount of mean body weight gained in the doses groups, the doses for females were then reduced to 1 000 and 4 000 respectively, and administration at the lowest doses was continued for 60 weeks. The time-weighted average doses for the female mice were either 2.315 or 5.741 ppm. matched controls consisted of 20 untreated mice of each sex. All surviving mice were killed at the end of administration of the test chemical.

Mean body weights of doses rats and mice of each sex were lower than those corresponding controls, and were dose related throughout the bioassay, except those of female rats during the first part of the bioassay. Mortality was dose related in the female rats, butwas not affected when the test chemical was administered to the male rats or the male and female mice. Survival at the end of the bioassay was 64% or grated in the dosed and control groups or rats and mice of each sex, and sufficient numbers of animals were at risk in all groups for the development of late-appearing tumors.

Transitional-cell carcinomas of the urinary bladder occurred at incidences that were dose related (p<0.001) in both male and femal rats, and in direct compisons the incidences of these tumors in the high-dose groups of each sex were significantly higher (p<0.001) than those in the corresponding controls (males: control 0/19, low-dose 0/46, high-dose 16/45; females : controls 0/18, low-dose 0/48, high dose 40/49). The possible mechanism by which these tumors were induced, such as calculi formation in the bladder or nitrosation of amines present in feed to a carcinogenic nitrosamine, is unknown.

Fibromas of the integumentary system occurred in male rats at incidences that were dose related (p=0.003), although in direct comparisons the incidences of these tumors in the individual dosed groups were not significantly higher than those in the control group (controls 1/20 = 5% ; low dose 1/50 = 2%; high dose 10/50 = 20%). The incidence of fibromas of the integumentary system in historical-control male F344 rats at this laboratory is 6/285 (2%). These results suggest an association of the fibromas in the male rats with administration of the test chemicals.

No tumors occurred in the mice of eihter sex at incidences that were significantly higher in the dosed groups than in the corresponding control groups. The onyl changes related to coumpound administration were chronic inflammatory lesions int he urinary bladders of dosed mice.

It is concluded that under the conditions of this bioassay, nitrosodiphenylamine was carcinogenic for both sexes of F344 rats, inducing transitional-cell carcinomas of the urinary bladder, but was not carcinogenic for B6C3F1 mice of either sex.

Preliminary study of the 2 -year carcinogenicity study (Innes 1969):

The results of this preliminary bioassay in mice did not clearly associate the incidence of any tumor with administration of nitrosodiphenylamine.

The tumorigenicity of selected pesticides and industrial compounds was tested by continuous oral administration to both sexes of two hybrid strains of mice, started at the age of 7 days.

Mice showed statistically significant increases in tumor incidence following gavage with 1000 mg/kg/day from day 7 -28 of age followed by dietary exposure to 3769 ppm until weeks 77 -79 of life.

Additional statistical evaluation and/or experimentation will be required before an interpretation can be made.

Carcinogenicity study in rat after oral administration (Druckrey 1967):

This study indicated a lack of tumorigenicity of the compound, after administration of 120 mg/kg per day of the compound for 700 days (approx. 24 months).

Carcinogenicity study in rat after diet exposure (Argus 1961):

No tumors were observed in the rats treated with nitrosodiphenylamine during 45 weeks in the diet.

DISCUSSION :

Feeding nitrosodiphenylamine mixed in solid food to rats gave rise to a significant incidence of urinary bladder tumors in rats and, therefore, the compound must be considered carcinogenic under the conditions of this study (NCI 1979). No significant increase of any tumors, compared with controls, was observed in mice (NCI 1979) ; however, the urinary bladder was clearly a target in mice for development of chemically related chronic inflammation. In addition, whereas the number of tumors and epithelial hyperplasia was small, it is consistent with the rest of the results that they were seen in treated animals only. While these tumors and hyperplasias may have evolved secondarily to the inflammatory lesions, with additional time, more inflammatory/proliferative lesions might have developed into flank neoplasia.

Some early studies reported no treatment-remated tumors in orally exposed rats (Argus 1961, Druckrey 1967). However, the bladder was not routinely examined in these studies. Indeed, the finding of transitional cell carcinomas in the bladders of rats given the high dose of nitrosodiphenylamine contrasts with previous reports of clonic feeding tests in rats (Druckrey 1967), which indicated a lack of tumorigenicity of the compound, aven after administration of 120 mg/kg per day of the compound for 700 days (approx. 24 months). In the NCI experiment, the dose of nitrosodiphenylamine given to the rats cannot be accurately quantified. However, assuming an intake of 20g of food per day by each 400 -g rat (values used by EFSA for older-rat), the highest dose used in NCI assay (4000 ppm) corresponded to 200 mg/kg. This dose, administered for 2 years, are higher than those given in Druckrey's experiments and may sufficiently different to account for the strong carcinogenic effet seen in NCI assay.