2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

Carcinogenicity

Description of key information

Based on the results of the Phase I, II and III studies that were performed to elucidate the MoA of PBO in mouse carcinogenicity, the mouse liver tumours are not considered relevant to humans due to qualitative differences between the two species. This MoA involves CAR activation and induction of replicative DNA synthesis in mouse hepatocytes. However, the MoA of PBO-induced mouse liver formation is qualitatively not plausible for humans. Specifically, PBO does not stimulate a proliferative response in cultured human hepatocytes, as it is observed in mouse. Therefore, it can be concluded that the occurrence of hepatic adenomas in mouse at high doses following life-time administration of PBO does not consist a cancer hazard for humans and classification for carcinogenicity is not proposed.

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:

1993

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 26.3 – 37.3g (males) and 21.3 – 28.0 g (females)
- Housing: All animals were housed individually in a stainless steel cages equipped with a wire-mesh floor
- Diet : ad libitum.
- Water: ad libitum
- Acclimation period: approximately three weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 40-70 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: January, 29, 1991 (date of arrive in the laboratory test) To: August, 19-22, 1992 ( Sacrifice date)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets were prepared weekly by direct addiction of PBO to ground rodent feed (Certified Rodent Chow 5002).
- Mixing appropriate amounts with: Certified Rodent Chow 5002.
- Storage temperature of food: Diets were stored in polyethylene containers at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The tability and the homogeneity of the test diets were verified by HPLC analysis.

Duration of treatment / exposure:

18 months

Frequency of treatment:

daily

Post exposure period:

none

Remarks:

Doses / Concentrations:
0 (two control groups) 30, 100, or 300 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

120/male/0; 60/male/30; 60/male/100; 60/male/300.
120/female/0; 60/female/30; 60/female/100; 60/female/300.

Control animals:

yes

Details on study design:

- Dose selection rationale: Due to the decreased body weights and hepatic changes including increased liver weight, hepatocellular necrosis and marked hepatocellular hypertrophy observed in the 90-day dose range-finding study, a target dosage of 1000 mg/kg/day was considered above the maximum tolerated dose. A target dosage of 300 mg/kg/day resulted in moderate liver changes in the 90-day study and was selected as the high dose for the oncogenicity study. A target dosage of 30 mg/kg/day in the 90-day study resulted in mild liver effects that were considered to be compensatory changes secondary to enzyme induction. Therefore a target dosage of 30 mg/kg/day as the low dose for the oncogenicity study. The mid dose group (a target dosage of 1000 mg/kg/day) was selected using a half log scale based upon the high dose.

Observations and examinations performed and frequency:

BODY WEIGHT: Individual body weights were recorded pretest and weekly during week 1-14 and every 2 weeks thereafter.

FOOD CONSUMPTION: Individual food consumption was recorded weekly during week 1-14 and every 2 weeks thereafter.

WATER CONSUMPTION: Not recorded

CLINICAL SIGNS: Animals were observed twice daily for mortality. Daily observations were conducted to assess overt toxicity. Weekly detailed observations were conducted to report clinical signs, body weights and food and compound consumption (the letter ones fortnightly from week 14 onwards).

OPHTHALMOSCOPIC EXAMINATION: Not reported
HAEMATOLOGY: Yes
- Number of animals: 10 animals/sex/high dose and control groups
- Time points: At week 52 and prior to the final sacrifice
- Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, MCV, MCH, MCHC

CLINICAL CHEMISTRY:No

URINALYSIS: No

PATHOLOGY: Yes

Sacrifice and pathology:

ORGAN WEIGHTS: Yes
- from: all surviving animals at terminal sacrifice
- Organs: Liver, kidneys, testes, , spleen, brain, heart

HISTOPATHOLOGY: Yes
- from: high dose group and controls at terminal sacrifice
- Organs: Brain, spinal cord, pituitary, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines, liver, pancreas, kidneys, adrenals, spleen, heart, trachea, lungs, aorta, gonads, uterus, female mammary gland, prostate, urinary bladder, gall bladder, lymph nodes, peripheral nerve, bone marrow, skin, eyes, skeletal muscle,
- from: 30 and 100 ppm, groups
- Organs: Lungs, liver, kidneys, gross lesions
Histopathology was performed on all gross lesions.

Statistics:

The data for quantitative continuous variables were intercompared for the 3 treatment groups and the 2 control groups by use of Levene’s test for equality of variances, analysis of variance (ANOVA), and t-tests.
Non parametric data were statistically evaluated using Kruskal-Wallis’ test followed by Mann-Whitney’s U-test. Incidence data were compared using Fisher’s Exact test.
New statistical analyses were conducted by Endura on the male mice groups (Fisher exact test, Peto test. (See the table "STATISTICAL ANALYSIS OF TUMOURS - FISHER EXACT TEST" reported in "Any other information on results incl. tables").

Clinical signs:

no effects observed

Description (incidence and severity):

See "Details on results"

Mortality:

no mortality observed

Description (incidence):

See "Details on results"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See "Details on results"

Ophthalmological findings:

not examined

Description (incidence and severity):

See "Details on results"

Haematological findings:

no effects observed

Description (incidence and severity):

See "Details on results"

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

See "Details on results"

Urinalysis findings:

no effects observed

Description (incidence and severity):

See "Details on results"

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results"

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results"

Details on results:

BODY WEIGHT: In the high dose group the mean absolute body weight and weight gains were decreased throughout the study. (See the table "MEAN BODY WEIGHTS AT STUDY WEEK 78" reported in "Any other information on results incl. tables").

FOOD CONSUMPTION: Mean food consumption values were comparable between both control groups and the treated dosage level groups. (See the tables "FOOD CONSUMPTION MALES" and "FOOD CONSUMPTION FEMALES" attached in "backgorund material").
The average amount of ingested piperonyl butoxide for 78 weeks is reported in the table "AVERAGE AMOUNT OF INGESTED PIPERONYL BUTOXIDE FOR 78 WEEKS" reported in "Any other information on results incl. tables"

WATER CONSUMPTION: Not recorded.

CLINICAL SIGNS: There were not treatment related clinical signs of toxicity and no significant effects on mortality (mean survival time) and no increases in palpable masses. (See the table "SURVIVAL DATA IN MICE TREATED WITH PIPERONYL BUTOXIDE" reported in "Any other information on results incl. tables").

MACROSCOPIC INVESTIGATIONS: Treatment related gross lesions included increased size and colour of the liver and increase incidence of combine nodules and masses in the liver of males in the mid dose group and of both sexes of the high dose group.

OPHTHALMOSCOPIC EXAMINATION: Not done

HAEMATOLOGY: No effects

CLINICAL CHEMISTRY: No effects

URINALYSIS: Not done

ORGAN WEIGHTS: In the mid and high dose groups of both sexes there was a dose related increase in absolute and relative liver weights.
The absolute and relative liver weights in the low dose group males were slightly increased (< 10%). (See the table " ABSOLUTE AND RELATIVE LIVER WEIGHTS OF MICE TREATED WITH PIPERONYL BUTOXIDE" reported in "Any other information on results incl. tables").

HISTOPATHOLOGY: Of 300 male mice, 212 survived to terminal sacrifice at week 79. There was a non-significant trend towards better survival with increasing dose of the test article, namely, Control (combined) 40/60 (33%), LD 41/60 (31.7%), ID 44/60 (26.7%) and HD 47/60 (21.7%).
The incidence of hepatocellular adenomas was increased for males in the mid dose group and for both sexes in the high dose group. The adenomas observed in Piperonyl Butoxide treated mice was different to those observed in the control groups. Basophilic adenomas in the liver were increased for males in the mid dose group and for both sexes in the high dose group. While eosinophilic adenomas were increased in males of all treated groups there was no dose relation.
Hepatocellular hyperplasia was increased in the high dose group of both sexes. Hepatocellular hypertrophy was increased in the high dose group males with a trend towards an increase in the mid dose group of males and the high dose group of females.
Regarding hepatocellular hypertrophy, there was a dose-related increase in hepatocellular hypertrophy in male mice.
Other treatment related microscopic lesions were observed in the high dose group male animals and included haemorrhage of the liver and increased sinus erythrocytosis of the mesenteric lymph nodes. These findings were considered secondary to hepatocellular changes in these animals.
No evidence of a treatment relationship with the incidence of liver carcinomas.
No evidence of a treatment relationship with the incidence of liver adenomas in decedents. However, in terminally killed animals, and in all animals, there was a positive dose-related trend in incidence in adenomas (p<0.001) with significant increases evident at 300 mg/kg/day (p<0.001) and 100 mg/kg/day (p<0.01), but not at 30 mg/kg/day. (See the tables "GROUP INCIDENCE OF LIVER PATHOLOGY IN MICE TREATED WITH PIPERONYL BUTOXIDE ", "DISTRIBUTION OF HEPATOCELLULAR PATHOLOGY IN MICE TREATED WITH PBO" and "STATISTICAL ANALYSIS OF TUMOURS - FISHER EXACT TEST" reported in "Any other information on results incl. tables")
Table A6.7/02-4 and Table A6.7/02-5 and Table A6.7/02-5 with new statistical data.

Result (carcinogenicity): negative

Dose descriptor:

LOAEL

Remarks:

Equivalent to 104.4 mg/Kg bw/day in males and 105.7 mg/Kg bw/day in females

Effect level:

>= 100 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on hepatotoxicity including increased absolute and relative liver weights in both sexes, and eosinophilic adenomas, nodules and masses in the liver, hepatocellular hypertrophy in males.

Dose descriptor:

NOAEL

Remarks:

Equivalent to 32.0 mg/Kg bw/day in males and 31.8 mg/Kg bw/day in females

Effect level:

>= 30 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on the results observed from the study on the correlation between the increase of the adenomas and the dose of test material provide.

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on the results observed about the increase of malignant tumours. (The study shows that there was no statistically significant increase in malignant tumours, which also fell within historical control for this strain of rat).

The tables reported below have been extracted by the robust study summary 91N0134

MEAN BODY WEIGHTS AT STUDY WEEK 78
Dosage level (ppm)	Group Mean Body Weights (g)
	Male			Female
		C 1	C 2		C 1	C 2
Control 1	43.4	-	-0.69	37.1	0.00	0.27
Control 2	43.7	0.69	-	37	-0.27	0.00
30	44.2	1.81	1.13	36.7	-1.09	-0.82
100	43.6	0.46	-0.23	37.3	0.54	0.80
300	41.1^1) 2)	-5.60	-6.33	37.5	1.07	1.33

C1 - % difference from Control 1

C2 - % difference from Control 2

¹⁾significantly different from Control 1, p<0.05

²⁾significantly different from Control 2, p<0.05

AVERAGE AMOUNT OF INGESTED PIPERONYL BUTOXIDE FOR 78 WEEKS
Dose level (ppm)	Compound ingested (mg/Kg bw/day)
	Males	Females
0	0	0
30	32.0	31.8
100	104.4	105.7
300	318.6	305.5

SURVIVAL DATA IN MICE TREATED WITH PIPERONYL BUTOXIDE
Treatment ppm	male			female
	Number	Number sacrificed	Mean Survival time	Number	Number sacrificed	Mean Survival time
Control 1	60	44	527	60	41	516
Control 2	60	36	501	60	49	540
30	60	41	520	60	44	528
100	60	44	529	60	37	525
300	60	47	536	60	40	510

ABSOLUTE AND RELATIVE LIVER WEIGHTS OF MICE TREATED WITH PIPERONYL BUTOXIDE
Dose level (mg/kg bw/day)	0	0	30	100	300
Males
Mean final body weight (g)	42.8	44.8	43.6	43.2	41.9
Mean absolute liver weight (g)	2.560	2.639	2.817	3.037^a	3.037^bd
Mean relative liver weight (%)	6.060	5.870	6.492	7.038^c	10.130^bd
Females
Mean final body weight (g)	38.1	37.5	37.6	38.9	38.1
Mean absolute liver weight (g)	2.208	2.153	2.182	2.446^ad	2.657^bd
Mean relative liver weight (%)	5.822	5.772	5.779	6.308^c	6.955^bd

^aSignificantly different from the first control group (p< 0.05)

^bSignificantly different from the first control group (p< 0.01)

^cSignificantly different from the second control group (p< 0.05)

^dSignificantly different from the second control group (p< 0.01)

GROUP INCIDENCE OF LIVER PATHOLOGY IN MICE TREATED WITH PIPERONYL BUTOXIDE
Dose level (mg/kg bw/day)	0	0	30	100	300
Males
Basophilic adenomas	7	6	10	15	10
Eosinophilic adenomas	4	1	3	8	19
Mixed cell adenomas	0	1	2	2	5
Females
Basophilic adenomas	1	0	0	0	2
Eosinophilic adenomas	1	2	1	1	9
Mixed cell adenomas	0	0	0	0	1

DISTRIBUTION OF HEPATOCELLULAR PATHOLOGY IN MICE TREATED WITH PBO
Dose level (mg/kg bw/day)	0	0	30	100	300
Males
Mice examined	60	60	60	60	60
Mice with adenoma	8	7	13	21	25
Mice with carcinoma	3	3	2	2	5
Mice with both adenoma and carcinoma	0	0	1	0	5
Mice with hyperplastic foci	0	2	1	2	5
Mice with liver hypertrophy	6	11	11	16	43
Mice with hepatocellular necrosis	21	20	11	14	29
Females
Mice examined	60	60	60	60	60
Mice with adenoma	2	2	1	1	10
Mice with carcinoma	0	0	0	0	0
Mice with hyperplastic foci	0	0	0	1	4
Mice with liver hypertrophy	0	4	0	1	9
Mice with hepatocellular necrosis	6	13	13	17	7

STATISTICAL ANALYSIS OF TUMOURS - FISHER EXACT TEST
Group		1	2	3	4	5
Dosage level (mg/kg/day)		Control A	30 mg/kg	100 mg/kg	300 mg/kg	Control E	Trend
Liver: carcinoma: Males
	n	3	2	2	5	3
	%	5.00	3.33	3.33	8.33	5.00
	P	C	0.9318	0.9318	0.5683	C	0.2917
Liver: adenoma: Males
	n	8	13	21	25	7
	%	13.33	21.67	35.00	41.67	11.67
	P	C	0.1706	0.0010 ++	0.0000 +++	C	0.0000 +++
Liver: adenoma or carcinoma:Males
	n	11	15	23	30	10
	%	18.33	25.00	38.33	50.00	16.67
	P	C	0.3228	0.0046 ++	0.0000 +++	C	0.0000 +++

There were not treatment related clinical signs of toxicity and no significant 

effects on mortality (mean survival time). No increases in  palpable masses, no 

changes in food consumption and no effects on  haematological parameters were 

observed in this study.In the high dose  group the mean absolute body weight and 

weight gains were decreased  throughout the study.In the mid and high dose 

groups of both sexes there  was a dose related increase in absolute and relative

 liver weights. The  absolute and relative liver weights in the low dose group 

males were  slightly increased (< 10%).Treatment related gross lesions included

  increased size and colour of the liver and increase incidence of combine 

nodules and masses in the liver  of males in the mid dose group and of  both 

sexes of the high dose group.Histologically, the incidence of  hepatocellular 

adenomas was increased for males in the mid dose group and  for both sexes of 

the high dose group. The adenomas observed in Piperonyl  Butoxide treated mice 

were different to those observed in the control  groups. Basophilic adenomas in 

the liver were increased for males in the  mid dose group and for both sexes of 

the high dose group. While  eosinophilic adenomas were increased in males of all

 treated groups there  was no dose relation.Hepatocellular hyperplasia was 

increased in the high  dose group of both sexes. Hepatocellular hypertrophy was 

increased in the  high dose group males with a trend towards an increase in the 

mid dose  group of males and the high dose group of females.Other treatment 

related  microscopic lesions were observed in the high dose group male animals 

and  included haemorrhage of the liver and increased sinus erythrocytosis of 

the mesenteric lymph nodes. These findings were considered secondary to 

hepatocellular changes in these animals.

Conclusions:

MATERIALS AND METHODS
In an oncogenicity study according to OECD Guideline 451, CD-1 mice in groups of 60 per sex were administered Piperonyl Butoxide in the diet at target doses of 0, 30, 100 or 300 mg/kg bw/d for at least 78 weeks. Two control groups were included in this study. Detailed clinical observations, including examinations of palpable masses, were conducted weekly throughout the study. Body weights and food consumption were measured weekly for the first 14 weeks and every other week thereafter. Haematology was conducted on 10 animals per sex from the high dose group and the control groups at weeks 52 and 78 of the study.
Complete necropsies were performed and selected organs were weighed for all animals of the study.
Histopathology was performed on full sets of organs from all animals of the high dose and control groups. In addition liver, lungs, kidneys and all gross lesions from all animals of the low and mid dose group were examined histologically. Two peer reviews of the histopathology of the liver were performed.

RESULTS AND DISCUSSION
Of 300 male mice, 212 survived to terminal sacrifice at week 79. There was a non-significant trend towards better survival with increasing dose of the test article, namely, Control (combined) 40/60 (33%), LD 41/60 (31.7%), ID 44/60 (26.7%) and HD 47/60 (21.7%).
There were not treatment related clinical signs of toxicity and no significant effects on mortality (mean survival time). No increases in palpable masses, no changes in food consumption and no effects on haematological parameters were observed in this study.
In the high dose group the mean absolute body weight and weight gains were decreased throughout the study.
In the mid and high dose groups of both sexes there was a dose related increase in absolute and relative liver weights. The absolute and relative liver weights in the low dose group males were slightly increased (< 10%).
Treatment related gross lesions included increased size and colour of the liver and increase incidence of combine nodules and masses in the liver of males in the mid dose group and of both sexes of the high dose group.
Histologically, the incidence of hepatocellular adenomas was increased for males in the mid dose group and for both sexes of the high dose group. The adenomas observed in Piperonyl Butoxide treated mice were different to those observed in the control groups. Basophilic adenomas in the liver were increased for males in the mid dose group and for both sexes of the high dose group. While eosinophilic adenomas were increased in males of all treated groups there was no dose relation.
Regarding hepatocellular hypertrophy, there was a dose-related increase in hepatocellular hypertrophy in male mice.

Hepatocellular hyperplasia was increased in the high dose group of both sexes. Hepatocellular hypertrophy was increased in the high dose group males with a trend towards an increase in the mid dose group of males and the high dose group of females.
Other treatment related microscopic lesions were observed in the high dose group male animals and included haemorrhage of the liver and increased sinus erythrocytosis of the mesenteric lymph nodes. These findings were considered secondary to hepatocellular changes in these animals.
No evidence of a treatment relationship with the incidence of liver carcinomas.
No evidence of a treatment relationship with the incidence of liver adenomas in decedents. However, in terminally killed animals, and in all animals, there was a positive dose-related trend in incidence in adenomas (p<0.001) with significant increases evident at 300 mg/kg/day (p<0.001) and 100 mg/kg/day (p<0.01), but not at 30 mg/kg/day.
Conclusions for the combined incidence of liver carcinomas or adenomas are essentially the same as for liver adenomas.
Various historical control data from BRRC and CRL showed that hepatocellular adenomas are common findings in male CD-1 control mice. At BRRC they have been reported at levels between 6/60 and 13/60. Hepatocellular adenomas are benign tumours & considered to be of questionable relevance in mice as they do/did not affect survival.

CONCLUSION
Eosinophilic adenomas in Piperonyl Butoxide treated animals are believed to occur as an extreme response to chronic induction of mixed function oxidases (Butler et al (1998) Hum Exp Toxicol 17, 323 and Butler (1996) Toxicol Pathol 24, 484).
There was no statistically significant increase in malignant tumours, which also fell within historical control for this strain of rat.
New statistics confirm no evidence of any treatment relationship with liver carcinomas; this confirms the original report conclusion with up to date methods.
The increased liver weight of mid and high dose level mice of both sexes was consistent with observed hypertrophy in mid and high dose males and high dose level female mice. This in turn was consistent with sustained induction of hepatic mixed function oxidases.
Dose related trend for increased adenomas at 100 and 300mg/kg but not at 30mg/kg, which is the NOAEL. There was a non-significant trend for better survival with increasing dosage of PBO.
Hepatocellular adenomas are common findings in male CD-1 control mice. At BRRC they have been reported at levels between 6/60 and 13/60 in male mice. Hepatocellular adenomas are benign tumours & considered to be of questionable relevance for risk assessment as they do/did not affect survival.

NOAEL for carcinogenicity = 300 mg/Kg bw/day

LOAEL = 100 ppm (equivalent to 104.4 mg/Kg bw/day in males and 105.7 mg/Kg bw/day in females), based on hepatotoxicity together with increased absolute and relative liver weights in both sexes, and eosinophilic adenomas, nodules and masses in the liver, hepatocellular hypertrophy in males and high dose level females.

NOAEL = 30 ppm (equivalent to 32.0 mg/Kg bw/day in males and 31.8 mg/Kg bw/day in females)

Other conclusions:
In the top dose group (both sexes) there was an increased incidence of hepatocellular hyperplasia and necrosis which was seen in the high dose level males and mid-dose females..
Males seem to be slightly more sensitive to piperonyl butoxide oral administration. In the case of liver adenoma the increased incidence is characteristic of male mice.
Target organ: liver

RELIABILITY: 1
DEFICIENCIES: No

Endpoint conclusion

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Justification for classification or non-classification

Piperonyl Butoxide is not considered carcinogenic.

Additional information

Piperonyl butoxide, (PBO), a synergist for a wide range of insecticides, was assessed in a 78-week United States Environmental Protection Agency (EPA) guideline dietary study in CD-1 mice and was shown to increase the incidence of liver adenoma in male mice at dietary levels of 100 and 300 mg/kg/day and in female mice at a dietary level of 300 mg/kg/day (Hermansky and Wagner, 1993). In contrast, PBO did not increase the incidence of liver or any other tumours in male and female Sprague Dawley (SD) rats in an EPA Guideline study fed at dietary levels up to 500 mg/kg/day for 2 years (Graham, 1987). Short term tests for genotoxic potential have demonstrated that PBO is not a genotoxic agent, (Butler et al, 1996). It can therefore be concluded that the formation of liver tumours in the mouse is due to a non-genotoxic mode of action (MOA). For non-genotoxic chemicals, established MOAs for rodent liver tumour formation include activation of receptors such as aryl hydrocarbon, constitutive androstane receptor (CAR), and peroxisome proliferator-activated receptor-alpha (PPARα), cytotoxicity, hormonal perturbation, and porphyria, (Cohen, 2010). Analysis of in vitro and in vivo investigative studies with PBO using sodium phenobarbital (NaPB) as an extensively studied prototype for comparison (IARC, 2001, Whysner et al., 1996 Rencurel et al., 2006, Hosseinpour, 2006 and Elcombe et al 2014), indicated a PBlike MOA for mouse liver tumour formation that is primarily due to CAR activation. This mode of action in the mouse carcinogenicity study is considered to have no relevance to humans due to a qualitative species difference between the mouse and man, where for man, CAR receptor activation does not result in a proliferative response in the liver whereas it does in the case of mice, (Lake, 2009 and Elcombe et al., 2014). In consequence, it can be concluded that the occurrence of hepatocellular adenoma in mice at high dose levels following life-time administration of PBO does not reflect a cancer hazard for man.

(See attachment "white paper on PBO MoA_HRF_final" in the information panel).

Based on a thorough review of the available toxicology database, it is clear that PBO is a non-genotoxic agent that has resulted in a proliferation of benign hepatic changes in the mouse; its activity is restricted entirely to one tissue, the liver. The apparent oncogenicity in mouse liver (a species with a propensity to develop a high proportion of liver tumours with or without exposure to chemicals) is clearly related to the ability of PBO to induce hepatocellular damage at high doses and in turn initiate round necrosis and regenerative hyperplasia. These events would, over chronic periods of treatment, allow the fixing of any spontaneously occurring DNA alterations thereby giving rise to a phenotypically distinct population of pre-initiated cells which then progress to adenomata. In addition to these high dose effects PBO also induces a work load hypertrophy and an induction of specific enzymes of the cytochrome P450 dependent mixed function oxidases and the associated increased protein synthesis, both of these being threshold responses. These changes are reversible following cessation of exposure. The liver changes induced are benign and are eosinophilic in appearance (generally associated with induction of mixed function oxidases by compounds which cause smooth endoplasmic reticulum proliferation), and there is no statistically significant increase in carcinomata.

The strong weight of evidence indicates that PBO used as recommended is most unlikely to present a carcinogenic risk to man since oncogenic response in the animal model is a threshold response with a NOEL which allows a safety factor approach to be employed. This lack of carcinogenic risk to man is an opinion confirmed by its long and safe history of use over the last 40 years.

(See attachment "PBO Mouse Oncogenicity WoE A6.10 -02" in the information panel)