N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine

Carcinogenicity

Description of key information

The data from both chronic feeding studies (Monsanto Chemical Co. 1993, Monsanto Co. 1978) did not indicate a carcinogenic potential of 6PPD in rats. 

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

Study period:

1987 - 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: non GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

; early chronic feeding study; GLP not mentioned

Principles of method if other than guideline:

chronic feeding study

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

substance was incorporated into diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

729, 730, 731, 733, 735 or 737 days depending upon the date of sacrifice.

Frequency of treatment:

daily via diet.

Post exposure period:

No

Remarks:

Doses / Concentrations:
0 ppm (control)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
50 ppm
Basis:
nominal in diet
2.6 mg/kg/day (males) and 3.2 mg/kg/day (females)

Remarks:

Doses / Concentrations:
250 ppm
Basis:
nominal in diet
13.5 mg/kg/day (males) and 16.5 mg/kg/day (females)

Remarks:

Doses / Concentrations:
1500 ppm
Basis:
nominal in diet
84.8 mg/kg/day (males) and 109.5 mg/kg/day (females)

No. of animals per sex per dose:

At the beginning of the experiment 70 rats/sex/group were dosed with 6PPD. At 12 months of treatment approximately 20 rats/sex/group were sacrificed. After 24 months of treatment all survivors were sacrificed.

Control animals:

yes, concurrent no treatment

Clinical signs:

no effects observed

Description (incidence and severity):

There were no statistically significant differences among survivorship or early deaths in any dose group compared to controls.

Mortality:

no mortality observed

Description (incidence):

There were no statistically significant differences among survivorship or early deaths in any dose group compared to controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight was consistently lower in males and females at 1500 ppm (mean difference to control – 9.9% and -18.4%, respectively) and lower in females at 250 ppm (-5.4%).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was increased in males and females at 1500 ppm (mean difference to control +5.5% and +17.3%, respectively) and in females at 250 ppm (+4.1%).

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related ophthalmoscopy observations were reported after 12 months or at termination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Overall, hematologic observations indicate that 6PPD might induce a slight anemia in animals of the high dose groups. For further details see "details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

increased cholesterol levels

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no consistent observations in in urine analysis for any parameter measured.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Clinical observations were of the type commonly seen in lifetime rodent studies and no consistent patterns were evident. There was an increase in the incidence of poor condition and emaciation of the high dose females, but not males.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See details on results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details on results

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Equivocal; see details on results

Details on results:

Hematologic observations were reported at the high dose:
- Hemoglobin was slightly reduced in males compared to control animals by 7, 8, and 14% at the high dose at 3, 6, and 12 months, respectively. Slight reduction was observed in females of the high dose group at all time points (12, 17, 14, 10 and 17% at 3, 6, 12, 18, and 24 months, respectively).
- Hematocrit was slightly reduced in males compared to control animals by 5, 7, and 13% at the high dose at 3, 6, and 12 months, respectively. Slight reduction was observed in females in the high dose group at all time points (12, 11, 11, 8, and 17%% at 3, 6, 12, 18, and 24 months, respectively).
- Erythrocyte counts were not affected in males but slightly reduced (between 7 - 13%) in females compared to controls in the high dose group at 3, 6, 12, and 24 months.
- Platelet counts were slightly increased at the high dose group in males at a single point in time (14% at 3 months). In females a slight increase between 8 and 30% was observed at 3, 6, 12, and 24 months.
- Mean corpuscular volume was slightly reduced (between 3 – 8% compared to control) in the high dose group in males and females at 3, 6, 12 and 18 months, but not at termination.
- Mean corpuscular hemoglobin was slightly reduced (between 5 – 9% compared to control) in the high dose group in males and females at 3, 6, 12, and 18 months , but not at termination.
- Mean corpuscular hemoglobin concentration was slightly reduced in the high-dose group in males at termination (3%) and in females at 3 and 6 months (3 and 3%, respectively).
- Total and differential leucocyte counts were not affected at any dose group or at any time point.
Overall, hematologic observations indicate that 6PPD might induce a slight anemia in animals of the high dose groups. Body and liver weights are already affected at the mid dose groups. No histopathologic observations are reported for any blood forming organ; absolute and relative spleen weights are increased only in the high-dosed males at 12 months, but not at termination. Taken together the hematologic observations are considered slight, high-dose effects with no morphologic correlates.

Clinical chemistry:
Consistent statistically significant increased cholesterol levels were observed in males and females in the high dose groups at 6, 12, 18, and 24 months.
Other observations on clinical chemistry were reported in the high-dose groups (increased total protein, globulin and calcium) but the effects are generally slight and the reported observations are not consistent over the different time points.

Post mortem organ toxicity:
Kidneys:
Mean absolute and relative kidney weights were increased in the high-dose groups in males and females at 12 months, but not at termination.
Chronic nephropathy correlated with and increased incidence of irregularities of the kidney surface observed macroscopically in males and females at the high dose groups. The incidence of chronic nephropathy was similar between the control groups and the dose groups. However, the severity of chronic nephropathy increased among the high-dosed animals of both sexes sacrificed after 12 months and at termination.

Liver:
Mean absolute and relative liver weights were increased in males and females after 12 and 24 months in the high-dose groups and in the mid-dose groups at termination. Microscopic observations indicate increased incidence in pigment in the hepathocytes and reticuloendothelial cells and cytoplasmic vacuolization of the liver in females at the high dose. No effect was observed in males or females in the mid dose or males in the high dose.

Thyroid:
Follicular cell hyperplasia was slightly but not statistically significant increased among treated males. Slightly increased follicular carcinoma was observed in males of the mid and high dose group with the following low incidences:
0 ppm (control) 50 ppm 250 ppm 1500 ppm
Follicular carcinoma
Females 1/69 2/70 1/69 1/69
Males 0/70 0/69 2/70 3/69
Follicular adenoma
Females 0/69 1*/70 0/69 1/69
Males 3/70 2/69 3/70 3/69
*Animal with follicular carcinoma and adenoma.

Calculation of the Fisher Extract Test showed that the increased incidence of follicular cell carcinoma seen in males at the mid and high dose groups was not statistically significantly different from controls. Nevertheless, the incidences in males in the mid (2/69; 2.9%) and high dose (3/69; 4.4%) are slightly above the historical control incidences observed in that laboratory (4/501; 0.8%).

The mechanism of potential thyroid gland follicular carcinoma observed in chronic rodent studies of chemicals is discussed in the literature. The thyroid hormone from the thyroid gland helps to set the metabolic rate of cells throughout the body. Thyroid function and thyroid cell division are regulated by circulating T4, T3 and TSH concentrations. As a result of iodide deficiency, or exposure to certain chemicals, the thyroid responds by increasing the size (hypertrophy) and number (hyperplasia) of thyroid follicular cells to enhance hormone output. A chronic hypertrophy and hyperplasia is known to be a key factor for tumor development. Since thyroid hormones are metabolized by the liver, largely by conjugation reaction, increased liver activity is known to induce thyroid follicular hypertrophy and hyperplasia and might lead to increased thyroid tumors as a secondary effect (e.g. Assessment of Thyroid Follicular Cell Tumor, EPA, Risk Assessment Forum, EPA/630/R-97/002, March 1998).
Based on the above mentioned considerations the slightly increased incidence of thyroid follicular carcinoma observed in this study is probably a secondary effect primarily based on liver activation. Mean absolute and relative liver weights were increased in males and females after 12 and 24 months in the high-dose groups. Unfortunately, liver weight has not been investigated for all animals with thyroid follicular tumors. Liver weight data are available for the high dose group for one male with follicular carcinoma (animal no. 4019) and two males with follicular adenoma (no. 4008 and no. 4047). The absolute and relative organ weights of these 3 males were not only elevated compared to controls but also at the higher end within the high dose group. The 3 males in question were the animals with the highest absolute or relative liver weights in the entire study (liver/brain weight for males 4019, 4008, 4047: 10.67, 11.58, 11.82; for comparison: mean liver/brain weight in the high dose group: 9.85; mean liver/brain weight in the control group: 7.00).

Overall, follicular cell carcinoma of the thyroid observed after 6PPD application in one chronic study (1993) in SD-rats are considered to be equivocal, based on 1) low, statistically not significant incidence in males, 2) no increase in females, 3) no effect on thyroid in an earlier chronic study (1978) in another rat strain (Charles River CD). Data on animals for which follicular cell carcinoma and liver weight data are available indicate that both observations are correlated and the effects on the thyroid gland are most likely to be high dose, secondary effects caused by increased liver activity. Together with the evidence that humans may not be as sensitive quantitatively to thyroid cancer development from thyroid-pituitary disruption as rodents (EPA/630/R-97/002, March 1998), the high-dose observations are considered to be of limited relevance for human risk assessment. It can be concluded that it is unlikely 6PPD possess a carcinogenic risk for humans.

Lung:
An increased incidence of adhesions and nodules/masses of the lung was reported in males but not in females, but these observations are not supported by microscopic evaluation.

Spleen:
Mean absolute and relative spleen weights were increased in the high-dose males only at 12 months, but not at termination.

Dose descriptor:

NOEL

Effect level:

ca. 2.6 - ca. 3 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

>= 84.8 - 109.5 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: It can be concluded that it is unlikely that 6PPD possess a carcinogenic risk for humans.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

The NOEL of this oral chronic toxicity study is 50 ppm in males and females (2.6 mg/kg/day in males, 3.2 mg/kg/day in females), based on reduced body weight in females, increased food consumption in females and increased liver weight at 250 ppm in both sexes (13.5-16.5 mg/kg/day). At the high dose (1500 ppm; 84.8-109.5 mg/kg/day) liver histopathology in females and slight effects on hematology are observed in both sexes.

Executive summary:

The NOEL of this oral chronic toxicity study is 50 ppm in males and females (2.6 mg/kg/day in males, 3.2 mg/kg/day in females), based on reduced body weight in females, increased food consumption in females and increased liver weight at 250 ppm in both sexes (13.5-16.5 mg/kg/day). At the high dose (1500 ppm; 84.8-109.5 mg/kg/day) liver histopathology in females and slight effects on hematology are observed in both sexes.

The follicular cell carcinoma of the thyroid observed in this study are considered to be equivocal, based on 1) low, statistically not significant incidence in males, 2) no increase in females, 3) no effect on thyroid in an earlier chronic study (1978) in another rat strain (Charles River CD). Data on animals for which follicular cell carcinoma and liver weight data are available indicate that both observations are correlated and the effects on the thyroid gland are most likely to be high dose, secondary effects caused by increased liver activity. Together with the evidence that humans may not be as sensitive quantitatively to thyroid cancer development from thyroid-pituitary disruption as rodents (EPA/630/R-97/002, March 1998), the high-dose observations are considered to be of limited relevance for human risk assessment.

Overall It can be concluded that it is unlikely that 6PPD possess a carcinogenic risk for humans.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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 classification is required according to the classification criteria 67/548/EEEC and regulation no. 1272/2008 (GHS).

Additional information

Carcinogenicity: oral

In a two-year study (Monsanto Chemical Co. 1993) 6PPD was administered orally, via dietary admixture to Sprague Dawley rats at dose levels of 0, 50, 250, and 1500 ppm. At the beginning of the experiment 70 rats/sex/group were dosed with 6PPD. At 12 months of treatment approximately 20 rats/sex/group were sacrificed. The No-observed-effect-level (NOEL) of this oral chronic toxicity study for non-neoplastic toxicity is 50 ppm in males and females (2.6 mg/kg/day in males, 3.2 mg/kg/day in females), based on reduced body weight in females, increased food consumption in females and increased liver weight at 250 ppm in both sexes (13.5-16.5 mg/kg/day). At the high dose (1500 ppm; 84.8-109.5 mg/kg/day) liver histopathology in females and slight effects on hematology are observed in both sexes. After 24 months of treatment all survivors were sacrificed. Follicular cell hyperplasia was slightly but not statistically significant increased among treated males. Slightly increased follicular carcinoma was observed in males of the mid and high dose group with the following low incidences:

0 ppm (control); 50 ppm; 250 ppm; 1500 ppm

Follicular carcinoma:

Females1/69; 2/70; 1/69; 1/69

Males 0/70; 0/69; 2/70; 3/69

Follicular adenoma:

Females 0/69; 1*/70; 0/69; 1/69; *Animal with follicular carcinoma and adenoma.

Males 3/70; 2/69; 3/70; 3/69

Calculation of the Fisher Extract Test showed that the increased incidence of follicular cell carcinoma seen in males at the mid and high dose groups was not statistically significantly different from controls. Nevertheless, the incidences in males in the mid (2/69; 2.9%) and high dose (3/69; 4.4%) are slightly above the historical control incidences observed in that laboratory (4/501; 0.8%). The mechanism of potential thyroid gland follicular carcinoma observed in chronic rodent studies of chemicals is discussed in the literature. The thyroid hormone from the thyroid gland helps to set the metabolic rate of cells throughout the body. Thyroid function and thyroid cell division are regulated by circulating T4, T3 and TSH concentrations. As a result of iodide deficiency, or exposure to certain chemicals, the thyroid responds by increasing the size (hypertrophy) and number (hyperplasia) of thyroid follicular cells to enhance hormone output. A chronic hypertrophy and hyperplasia is known to be a key factor for tumor development. Since thyroid hormones are metabolized by the liver, largely by conjugation reaction, increased liver activity is known to induce thyroid follicular hypertrophy and hyperplasia and might lead to increased thyroid tumors as a secondary effect (e. g. Assessment of Thyroid Follicular Cell Tumor, EPA, Risk Assessment Forum, EPA/630/R-97/002, March 1998). Based on the above mentioned considerations the slightly increased incidence of thyroid follicular carcinoma observed in this study is probably a secondary effect primarily based on liver activation. Mean absolute and relative liver weights were increased in males and females after 12 and 24 months in the high-dose groups. Unfortunately, liver weight has not been investigated for all animals with thyroid follicular tumors. Liver weight data are available for the high dose group for one male with follicular carcinoma (animal no. 4019) and two males with follicular adenoma (no. 4008 and no. 4047). The absolute and relative organ weights of these 3 males were not only elevated compared to controls but also at the higher end within the high dose group. The 3 males in question were the animals with the highest absolute or relative liver weights in the entire study (liver/brain weight for males 4019, 4008, 4047: 10.67, 11.58, 11.82; for comparison: mean liver/brain weight in the high dose group: 9.85; mean liver/brain weight in the control group: 7.00).

Follicular cell carcinoma were not observed in an earlier chronic study on Charles River rats (Monsanto Co.,1978). In this two-year study 50 rats per sex and dose were fed with 0, 100, 300 or 1000 ppm 6PPD in the diet (approx. 0, 8, 23 and 75 mg/kg bw/day) (Monsanto Co.1978). Survival of treated animals was comparable to control animals. Chronic toxicity was indicated by depressed body weights, reduced erythrocyte counts, haemoglobin concentrations and hematocrit values in high dose animals (for more details see chapter repeated dose toxicity). Gross autopsy examination on animals did not reveal any adverse effects related to the test substance administered. In addition, microscopic examination of suspect neoplasms was conducted on all sacrificed animals and all animals that died during the study. ANOAEL of 300 ppm for systemic toxicity (ca. 23 mg/kg bw/day) is suggested for this study.No differences were noted between test and control rats as to the organ system, type or classification of neoplasms. The spectrum of neoplasms observed compared favourably to historical control data.

 

Overall, follicular cell carcinoma of the thyroid observed after 6PPD application in one chronic study (Monsanto Chemical Co. 1993) in SD-rats are considered to be equivocal, based on 1) low, statistically not significant incidence in males, 2) no increase in females, 3) no effect on thyroid in an earlier chronic study (1978) in another rat strain (Charles River CD). Data on animals for which follicular cell carcinoma and liver weight data are available indicate that both observations are correlated and the effects on the thyroid gland are most likely to be high dose, secondary effects caused by increased liver activity. Together with the evidence that humans may not be as sensitive quantitatively to thyroid cancer development from thyroid-pituitary disruption as rodents (EPA/630/R-97/002, March 1998), the high-dose observations are considered to be of limited relevance for human risk assessment. It can be concluded that it is unlikely 6PPD possesses a carcinogenic risk for humans.

Justification for selection of carcinogenicity via oral route endpoint:
Most recent and comprehensive study.