p-benzoquinone dioxime

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

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

not specified

GLP compliance:

no

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Common rodent species historically used in carcinogenicity assays; note that mice of strain: B6C3F1 (male/female) were additionally dosed in parallel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: two animal species, Fischer 344 rats and B6C3F1 mice, used – sourced from Recognised Supplier.
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: ca. 6 weeks old (rats) ; ca. 6 weeks old mice.
- Weight at study initiation:
- Fasting period before study:
- Housing: Group housing four or five by sex, polycarbonate cages and stainless steel mesh. Filter paper was changed every 2 weeks. Clean cages and bedding with hardwood chip bedding renewed twice weekly.
- Diet (e.g. ad libitum): ad libitum, (test item treated/untreated, as applicable) Lab-Blox basal diet
- Water (e.g. ad libitum): ad libitum. Acidulated water..
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY: Information provided in the full study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 26 °C
- Humidity (%): 45 - 55
- Air changes (per hr): Filtered, 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 16 hours dark / 8 hours light

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency):
The mixture was prepared once weekly.
- Mixing appropriate amounts with (Type of food):
The basal laboratory diet for both dosed and control animals consisted of Wayne Lab-Blox meal. Test item was administered to the dosed animals as a component of the diet. The test item was removed from its container and a proper amount was blended with an aliquot of the feed using a mortar and pestle. Once visual homogeneity was attained, the mixture was placed in a 6 kg capacity Patterson-Kelley standard model twin-shell stainless steel V-blender along with the remainder of the feed to be prepared. After 20 minutes of blending, the mixtures were placed in double plastic bags and stored in the dark at 4°C. The mixture was prepared once weekly.
- Storage temperature of food:
stored in the dark at 4°C

- Other: Dosed feed preparations containing 1500 and 375 ppm were analysed spectrophotometrically. Mean result immediately after preparation was 97 percent of theoretical (ranging from 96 to 98 percent). After 10 days at ambient room temperature the mean result was 78 percent of theoretical (ranging from 73 to 82 percent). Applicant assessment indicates: the test item was stable for ca. 7 days (70 - 110% nominal) under the conditions employed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosed feed preparations containing 1500 and 375 ppm were analysed spectrophotometrically. Mean result immediately after preparation was 97 percent of theoretical (ranging from 96 to 98 percent). After 10 days at ambient room temperature the mean result was 78 percent of theoretical (ranging from 73 to 82 percent). Applicant assessment indicates: the test item was stable for ca. 7 days (70 - 110% nominal) under the conditions employed.

Duration of treatment / exposure:

2 years (or 104 weeks) treatment and 1 week observation

Frequency of treatment:

daily

Post exposure period:

1 week

No. of animals per sex per dose:

20 per control
50 per low dose (rat: 375 ppm and mouse: 750 ppm)
50 per high dose (rat: 750 ppm and mouse: 1500 ppm)

Control animals:

yes, plain diet

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Statistical analysis was perfomed not limited to:
1. Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958) and are presented in the report.
2. Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not statistically censored. Statistical analyses for a possible dose-related effect
on survival used the method of Cox (1972) when testing two groups for equality and used Tarone's (1975) extensions of Cox's methods when testing a dose-related trend. One-tailed P-values have been reported for all tests except the departure from linearity test, which is only reported when its two-tailed P-value is less than 0.05.
3. The incidence of neoplastic or nonneoplastic lesions is given as the ratio of the number of animals bearing such lesions at a specific anatomic site (numerator) to the number of animals in which that site was examined (denominator). Additional considerations were given.
4. one-tailed Fisher exact test (Cox, 1970, pp. 48-52) was used to compare the tumor incidence of a control group to that of a group of treated animals at each dose level.
5. Bonferroni inequality (Miller, 1966, pp. 6-10) requires that the P-value for any comparison be less than or equal to 0.05/k.
6. The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971, pp. 362-365), was also used when appropriate.
7. time-adjusted analysis was applied when numerous early deaths resulted from causes that were not associated with the formation of tumors.
8. life-table methods were used to analyze the incidence of tumors. Curves of the proportions surviving without an observed tumor were computed as in Saffiotti et al. (1972).
9. The approximate 95 percent confidence interval for the relative risk of each dosed group compared to its control was calculated from the exact interval on the odds ratio (Gart, 1971).

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

The Tarone test for association between dosage and mortality was not significant for either males or females.
Females: 78 percent (39/50) of the high dose, 78 percent (38/49) of the low dose, and 90 percent (18/20)
Males: 70 percent (35/50) of the high dose, 78 percent (39/50) of the low dose, and 85 percent (17/20) of the controls survived on test until the termination of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dose-related mean body weight depression was apparent in both male and female rats at the dose levels tested.

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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed a variety pf neoplasms in male and female rats with the highest incidence in the urinary, endocrine and reproductive systems. Except for the urinary system the distribution of neoplasms was judged to be random among dosed and control animals, and the neoplasms observed were of the type commonly observed in aging Fischer 344 rats.

Treatment related effects observed appeared confined to females in the urinary tract.

Other effects:

no effects observed

Relevance of carcinogenic effects / potential:

Within female and male rats:
1. transitional-cell papilloma 0/45, 2/46, 1/43, and 4/44 in the low dose males, high dose males, low dose females, and high dose females, respectively;
2. transitional-cell carcinoma 0/45, 0/46, 1/43, and 7/44 in the low dose males, high dose males, low dose females, and high dose females, respectively;
3. squamous-cell carcinomas 0/45, 0/46,-1/43, and 0/44 in the low dose males, high dose males, low dose females, and high dose females, respectively

Cochran-Armitage test for the female rats indicated a significant positive association between the concentrations administered and the incidences of these tumors when combined. The high dose to control Fisher exact comparison for females was also significant.

In female mice there was a significant positive association between chemical administration and the incidence of hepatocellular neoplasms. However, the Fisher exact comparisons did not achieve statistical significance. The authors concluded that this was not conclusive to relate to a test item effect given the historical range for the B6C3F1 mouse.

Critical effects observed:

yes

Lowest effective dose / conc.:

375 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Under the conditions of this study, there was a positive dose-related association between test item administration and neoplastic effects females in the urinary tract.

Executive summary:

The study was performed as a 104 week chronic carcinogenicity study using methods equivalent or similar to the requirements of OECD Guideline 453, to evaluate the potential carcinogenicity of the test item via the dietary route. Chronic toxicity tests were- conducted with both Fisher 344 rats and B6C3F1 mice. F344 male/female rats were distributed among two treatment groups of low: 375 ppm and high: 750 ppm, each consisting of fifty males and fifty females. B6C3F1 strain male/female mice were distributed among two treatment groups of low: 750 ppm and high: 1500 ppm, each consisting of fifty males and fifty females. Negative controls consisted of twenty rats, and/or mice untreated with plain diet. The test item was administered for 102-weeks through basal laboratory diet supplied ad libitum. Observation continued for one week post administration. Rats and mice were necropsied post termination. The basal laboratory diet for both dosed and control animals consisted of Wayne Lab-Blox meal. Test item was administered to the dosed animals as a component of the diet. The test item was removed from its container and a proper amount was blended with an aliquot of the feed using a mortar and pestle. Once visual homogeneity was attained, the mixture was placed in a 6 kg capacity Patterson-Kelley standard model twin-shell stainless steel V-blender along with the remainder of the feed to be prepared. After 20 minutes of blending, the mixtures were placed in double plastic bags and stored in the dark at 4°C. The mixture was prepared once weekly. The report indicated that dosed feed preparations containing 1500 and 375 ppm were analysed spectrophotometrically. Mean result immediately after preparation was 97 percent of theoretical (ranging from 96 to 98 percent). After 10 days at ambient room temperature the mean result was 78 percent of theoretical (ranging from 73 to 82 percent). Applicant assessment indicates: the test item was stable for ca. 7 days (70 - 110% nominal) under the conditions employed.

There were no significant positive associations between the concentrations of test item administered and mortality in rats or mice of either sex. Adequate numbers of animals in all groups survived sufficiently long to be at risk from late-developing tumors. Distinct dose-related mean body weight depression was observed among rats and slight mean body weight depression, relative to controls, was observed among mice, indicating that the dosages of test item administered may have approximated the maximum tolerated concentrations. Tumors of the urinary bladder were observed only in dosed rats. For female rats, there was a significant positive association between concentration administered and the incidences of a combination of urinary bladder neoplasms. The high dose to control Fisher exact comparison was also significant for these tumors in female rats. No other test item related neoplasms were observed in male rats or mice of either sex or were considered as relevant. In female mice there was a significant positive association between chemical administration and the incidence of hepatocellular neoplasms. However, the Fisher exact comparisons did not achieve statistical significance. The authors concluded that this was not conclusive to relate to a test item effect given the historical range for the B6C3F1 mouse.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

18.75 mg/kg bw/day

Study duration:

chronic

Species:

rat

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

Mode of Action Analysis / Human Relevance Framework

Not applicable. Mode of action is inconclusive based on current dataset.

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for carcinogenicity: category 2: H351: Suspected of causing cancer

Applicant assessment indicates: Metabolism following oral and inhalation administration is mainly through phase I and II enzymes in the liver and lung. The main phase I reactions would most like be an oxidative reaction followed by hydrolysis reaction, this would result into production of benzoquinone and nitro derivative and potentially reactive radical species.  The benzoquinone is expected to undergo redox cycling by NAD(P)H-quinone oxioreductase or carbonyl reductase forming hydroquinone, semiquinione and reactive oxygen species (ROS). Phase II reaction would potentially involve conjugation of hydroquinone with sulphate and glucuronic acid by the enzymatic activity of sufotransferase and glucuronosyl transferase, respectively. In addition to this, hydroquinone can also undergo redox cycling forming semiquinione which can be further conjugated with glutathione under the influence of glutathione s-transferase and the glutathione conjugate can be further metabolized to cysteine conjugates and mercapturic acids by enzymes such as N-acetyltransferease. The semiquinione formed can also undergo further redox cycling in presence of molecular oxygen, resulting in ROS, including superoxide anion (O2.-) and H2O2. The generation of ROS is supported by the neoplasm observed in the kidney, liver, lung and urinary bladder, elevated ROS are known to initiate cancer. The substance is expected to undergo redox cycling reaction by NAD(P)H-quinone oxidoreductase or carbonyl reductase producing metabolic conjugates and reactive oxygen species, the conjugates are erected via urine and bile while the ROS can bind into protein triggering cascade of events such DNA strand break. However, the substance did not induce any DNA damage in unscheduled DNA synthesis – OECD 486 nor was it mutagenic in micronucleus studies conducted according to OECD 474. The substance and or its metabolites could be considered as non-genotoxic carcinogens acting through cell proliferation as demonstrated by the positive results of renal carcinoma - OECD 453. Based on data obtained on cancer bioassays, an increase in proliferative response caused by chemicals/metabolites induced cytotoxicity which appears to play a role in the initiation of neoplasia [reference: Waris G and Ahsan H (2006)]. Since the renal carcinoma is only observed in rat of both sexes, the effect can be considered accumulation of α2u-globulin, which is known to have some association with renal carcinoma. The study did not indicate any measurement of α2u-globulin and we cannot rule out its association with the observed effects.

At high doses, the substance is not well tolerated as demonstrated by mortalities and overt toxicities noted in OECD 401 and OECD 453, hence the classification of Acute toxicity category 4 and CMR category 2 according to regulation No 1272/2008 (CLP). However, the urinary bladder tumour observed was sex and species specific to female rats only, this may little relevance to risk assessment in humans considering such effects was not observed in mice.

Additional information

eq. or similar to OECD TG 453, 1979: The study was performed as a 104 week chronic carcinogenicity study using methods equivalent or similar to the requirements of OECD Guideline 453, to evaluate the potential carcinogenicity of the test item via the dietary route. Chronic toxicity tests were- conducted with both Fisher 344 rats and B6C3F1 mice. F344 male/female rats were distributed among two treatment groups of low: 375 ppm and high: 750 ppm, each consisting of fifty males and fifty females. B6C3F1 strain male/female mice were distributed among two treatment groups of low: 750 ppm and high: 1500 ppm, each consisting of fifty males and fifty females. Negative controls consisted of twenty rats, and/or mice untreated with plain diet. The test item was administered for 102-weeks through basal laboratory diet supplied ad libitum. Observation continued for one week post administration. Rats and mice were necropsied post termination. The basal laboratory diet for both dosed and control animals consisted of Wayne Lab-Blox meal. Test item was administered to the dosed animals as a component of the diet. The test item was removed from its container and a proper amount was blended with an aliquot of the feed using a mortar and pestle. Once visual homogeneity was attained, the mixture was placed in a 6 kg capacity Patterson-Kelley standard model twin-shell stainless steel V-blender along with the remainder of the feed to be prepared. After 20 minutes of blending, the mixtures were placed in double plastic bags and stored in the dark at 4°C. The mixture was prepared once weekly. The report indicated that dosed feed preparations containing 1500 and 375 ppm were analysed spectrophotometrically. Mean result immediately after preparation was 97 percent of theoretical (ranging from 96 to 98 percent). After 10 days at ambient room temperature the mean result was 78 percent of theoretical (ranging from 73 to 82 percent). Applicant assessment indicates: the test item was stable for ca. 7 days (70 - 110% nominal) under the conditions employed.

There were no significant positive associations between the concentrations of test item administered and mortality in rats or mice of either sex. Adequate numbers of animals in all groups survived sufficiently long to be at risk from late-developing tumors. Distinct dose-related mean body weight depression was observed among rats and slight mean body weight depression, relative to controls, was observed among mice, indicating that the dosages of test item administered may have approximated the maximum tolerated concentrations. Tumors of the urinary bladder were observed only in dosed rats. For female rats, there was a significant positive association between concentration administered and the incidences of a combination of urinary bladder neoplasms. The high dose to control Fisher exact comparison was also significant for these tumors in female rats. No other test item related neoplasms were observed in male rats or mice of either sex or were considered as relevant. In female mice there was a significant positive association between chemical administration and the incidence of hepatocellular neoplasms. However, the Fisher exact comparisons did not achieve statistical significance. The authors concluded that this was not conclusive to relate to a test item effect given the historical range for the B6C3F1 mouse.