ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

Carcinogenicity

Description of key information

OECD 453, rat, oral: NOAEL carcinogenicity = 210 mg/kg bw/day, no indication of carcinogenic effects

OECD 451, mouse, oral: NOAEL carcinogenicity = 372 mg/kg bw/day, no indication of carcinogenic effects

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Feb 1997 - 28 Aug 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

Current version adopted in 2018

Deviations:

yes

Remarks:

Sacrifice of the high dose group was ahead of schedule due to accidental overdosing in Week 22. Subsequently another study with this dose level was conducted.

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

Guideline in place during study conduct: adopted in 1981

Deviations:

yes

Remarks:

Sacrifice of the high dose group was ahead of schedule due to accidental overdosing in Week 22. Subsequently another study with this dose level was conducted.

Qualifier:

according to guideline

Guideline:

EU Method B.32 (Carcinogenicity Test)

Version / remarks:

Adopted in 1987

Deviations:

yes

Remarks:

Sacrifice of the high dose group was ahead of schedule due to overdosing in Week 22. Subsequently another study with this dose level was conducted.

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Version / remarks:

Adopted in 1984

Qualifier:

according to guideline

Guideline:

other: JMAFF (Oncogenicity Study, Carcinogenicity)

Version / remarks:

Adopted in 1985

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, Sulzfeld, Germany
- Age at receipt: approx. 5 - 6 weeks
- Mean body weight at study initiation: males: 24.2 g, females: 21.1 g
- Housing: individually in Macrolon (type II) cages with soft wood granulate bedding
- Diet: powdered laboratory rodent diet, Ssniff (V1530)-ground, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY: Contaminants for both, food and water were periodically analysed and the results are archived. None of the contaminants was present at a level that was considered likely to influence the outcome or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes: air-conditioned room
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Feb 1997 To: 28 Aug 1998

Route of administration:

oral: feed

Vehicle:

other: powdered laboratory rodent diet

Remarks:

Ssniff (V1530)-ground

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): In 1 - 4 weekly intervals, stored in closed plastic containers at ambient temperature. Pre-test analysis confirmed the stability of the test substance in food over 35 days at 6 and 2500 ppm.
- Mixing appropriate amounts: The required amount of test material and a small amount of diet were mixed. This mix was then blended with the remaining diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From the final mixtures homogeneity and actual active ingredient content of the test substance in the diet was determined by HPLC, before start of study, at Week 24, 36, 48, 60 and 72. The results were between 81 and 112% of the nominal concentrations, hence, within the acceptable range of 80 to 120% of nominal.
Analysis of the by mistake 10-fold overdosed 2500 ppm mixture used in Week 22 showed a nominal concentration of 1084% of nominal.

Duration of treatment / exposure:

18 months: control, 12.5, 125, and 1250 ppm
23 weeks: 2800 ppm
Due to an error the 2500 ppm dose group was treated with a 10-fold higher dose for approx. 1 week (Week 22). This caused intercurrent deaths and clinical symptoms of toxicity. Consequently this dose group was terminated in Week 23.

Frequency of treatment:

Continuously

Dose / conc.:

12.5 ppm

Remarks:

actual test substance intake: males: 1.67 mg/kg bw/day, females: 2.08 mg/kg bw/day, combined sexes: 1.88 mg/kg bw/day

Dose / conc.:

125 ppm

Remarks:

actual test substance intake: males: 16.61 mg/kg bw/day, females: 19.88 mg/kg bw/day, combined sexes: 18.24 mg/kg bw/day

Dose / conc.:

1 250 ppm

Remarks:

actual test substance intake: males: 169.63 mg/kg bw/day, females: 202.49 mg/kg bw/day, combined sexes: 193.42 mg/kg bw/day

Dose / conc.:

2 500 ppm

Remarks:

actual test substance intake until Week 22: males: 402.88 mg/kg bw/day, females: 500.85 mg/kg bw/day, combined sexes: 451.87 mg/kg bw/day; dose group terminated in Week 23 due to dosing error in Week 22.

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of a previous 90-day repeat dose toxicity study in mice (M-142011-01-1). In this study, dietary administration of the test substance at 2500 ppm resulted in centrilobular hepatocyte enlargement and vacuolation in males. Increased centrilobular fat deposition was also seen in the liver of both sexes. In addition, there was a 26% reduction in absolute kidney weight in males but a 25% increase in both absolute and relative kidney weights in females. Therefore, this dose level was expected to approximate to the maximum tolerated dose (MTD) in this oncogenicity study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Behaviour and general health condition were observed twice daily (on weekends and public holidays once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Monthly, the animals were examined for neurological disturbances, impairment of dental growth and changes in the eyes and oral mucosa. Moreover, each animal was palpated for skin masses monthly for the first 6 months of treatment and afterwards twice monthly.

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly from the start of treatment (Day 1).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Individually and weekly combined with the body weight examination.
- Compound intake was calculated on the basis of mean weekly relative food consumption and nominal dose levels.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Monthly (see 'Detailed clinical observations')
- How many animals: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 12 months and prior to necropsy.
- Animals fasted: No
- How many animals: All
- Parameters checked: Haematocrit, Haemoglobin, Erythrocyte count, Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Platelet count, Reticulocyte count, Total leucocyte count, and Differential blood count.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- List of organs: Adrenals, aorta, bone marrow (sternum), brain (with medulla oblongata), caecum, cartilage-bone junction, colon, diaphragm, duodenum, epididymides, ethmoid turbinals (head, middle ear), eyes, femur joint, gall bladder, Harderian gland, heart, ileum, jejunum, kidneys, lacrimal gland, larynx, liver, lungs, lymph nodes (mandibular, iliac and mesenteric), mammary gland, muscle (skeletal), nerves (optic and sciatic), oesophagus, oviducts, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (mandibular, sublingual and parotid), seminal vesicles, skin, spinal cord (3 levels), spleen, stomach, testes, thymus, thyroid (with parathyroids), tongue, trachea, urinary bladder, uterus, vagina, and any other tissue showing macroscopic abnormalities.
- Organ weights: Adrenals, brain, heart, kidneys, liver, ovaries with oviducts, spleen, and testes.

HISTOPATHOLOGY: Yes
Histopathology was conducted on organs/tissues indicated above from all animals.

Statistics:

For statistical analysis of in-life findings, haematology and organ weights the 2-tailed significance was evaluated by comparing the treated group with control.

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment-related changes in general health and behaviour (clinical signs of toxicity), eyes, teeth and oral mucosa, as well as during palpation of skin for nodules and masses at dose levels up to 1250 ppm were not observed.
Due to accidental overdosing in the 2500 ppm dose group, effects of this dose level are considered in the subsequent study, study entry "key, 1999, M-185504-01-1, mouse, part B, RL1".

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no treatment-related effects on survival or mortality rates in females up to the 1250 ppm dose group. In males of the 1250 ppm dose group mortality was slightly increased (see Table 2 under "Any other information on results incl. tables").

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Intermittently, slightly but statistically significantly higher body weights occurred in all treated male groups. Additionally, slightly higher overall mean body weight gains in males from the 125 and 1250 ppm groups were calculated. However, since there was no clear dose-response relationship and the differences were only marginal, they are considered to be incidental and of no biological relevance.
The statistically significant increase (2500 ppm on Day 8) and decreases (1250 ppm on Day 155, 204 and 484) in females are considered incidental and of no biological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Statistical evaluation of the haematological parameters examined after the scheduled sacrifice revealed slight but statistically significant changes of some parameters at 125 and 1250 ppm. However, none of the parameters was dose-dependently changed and all changes were within the typical range of this mouse strain. Therefore, they were considered not treatment related and not toxicologically relevant.

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:

no effects observed

Description (incidence and severity):

At 1250 ppm, absolute kidney weights were decreased (-7%) in females to a statistically significant degree. Relative kidney weights showed a comparable tendency but without statistical significance. Relative adrenal weights were statistically significantly reduced (-14.2%) in males. A statistically significant decrease of heart weights (absolute -10.3% and relative -9.7%) was calculated in females.
However, in the absence of any histopathological correlation in these organs the slight organ weight decreases were considered to be of no toxicological importance.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Relevance of carcinogenic effects / potential:

No carcinogenic effects found.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Remarks on result:

not determinable because of methodological limitations

Remarks:

The NOAEL for carcinogenicity is discussed in a subsequent study (M-185504-01-1). In the present study, no carcinogenic effect was observed after dietary exposure of mice up to 1250 ppm for 18 months. In the subsequent study, mice were exposed to 2500 ppm for 18 months.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

1 250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased mortality rate

Remarks on result:

other: actual test substance intake: 193.42 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

125 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake: 18.24 mg/kg bw/day

Key result

Critical effects observed:

no

Table 1: Group mean test substance intake (mg/kg bw/day)

Weeks		Dose level (ppm)
		12.5	125	1250	2500*
1-80	Males	1.67	16.60	169.63	402.88
	Females	2.08	19.88	202.49	500.85
	Combined sexes	1.88	18.24	193.42	451.87

* Weeks 1 - 22. High dose level mentioned in the text or for the effect levels is taken from Part B.

 

Table 2: Mortality

Week	Dose level (ppm)
	0		12.5		125		1250		2500*
	m	f	m	f	m	f	m	f	m	f
1-26	0	0	0	1	0	0	2	0	7	20
27-52	0	1	1	2	0	3	1	1
53-80	5	9	6	11	7	11	10	12
Total deaths	5	10	7	14	7	14	13	13
% mortality	10	20	14	28	14	28	26	26	14	40

* Week 1 - 23

Conclusions:

Under the conditions of the present study, after dietary administration of the test substance at dose levels up to 1250 ppm for 80 weeks no carcinogenic effect in CD-1 mice was observed.
A slightly increased mortality rate in male animals was observed at 1250 ppm.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jul 1997 - 02 Feb 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

Current version adopted in 2018

Deviations:

yes

Remarks:

Study was conducted with only one dose level due to overdosing in a preceding study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

Guideline in place during study conduct: adopted in 1981

Deviations:

yes

Remarks:

Study was conducted with only one dose level due to overdosing in a preceding study.

Qualifier:

according to guideline

Guideline:

EU Method B.32 (Carcinogenicity Test)

Version / remarks:

Adopted in 1987

Deviations:

yes

Remarks:

Study was conducted with only one dose level due to overdosing in a preceding study.

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Version / remarks:

Adopted in 1984

Qualifier:

according to guideline

Guideline:

other: JMAFF (Oncogenicity Study, Carcinogenicity)

Version / remarks:

Adopted in 1985

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, Sulzfeld, Germany
- Age at receipt: approx. 5 - 6 weeks
- Mean body weight at study initiation: males: 24.8 g, females: 20.4 g
- Housing: individually, in Macrolon (type II) cages with soft wood granulate bedding
- Diet: powdered laboratory rodent diet, Ssniff (V1530)-ground, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY: Contaminants for both, food and water were periodically analysed and the results are archived. None of the contaminants was present at a level that was considered likely to influence the outcome or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes: air-conditioned room
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30 Jul 1997 To: 02 Feb 1999

Route of administration:

oral: feed

Vehicle:

other: powdered laboratory rodent diet

Remarks:

Ssniff (V1530)-ground

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): In 1 - 4 weekly intervals, stored in closed plastic containers at ambient temperature. Pre-test analysis confirmed the stability of the test substance in food over 35 days at 2500 ppm.
- Mixing appropriate amounts: The required amount of test material and a small amount of diet were mixed. This mix was then blended with the remaining diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From the final mixtures homogeneity and actual active ingredient content of the test substance in the diet was determined by HPLC, before start of study, at Week 5, 9, 13, 25, 37, 49, 61 and 73. The results were between 91 and 107% of the nominal concentrations, i.e. within the acceptable range of 80 to 120% of nominal.

Duration of treatment / exposure:

18 months

Frequency of treatment:

Continuously

Dose / conc.:

2 500 ppm

Remarks:

actual test substance intake: males: 336.85 mg/kg bw/day, females: 407.28 mg/kg bw/day, combined sexes: 372.07 mg/kg bw/day

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The 2500 ppm dose level was selected on the basis of a previous 90-day repeat dose toxicity study in mice (M-142011-01-1). The present study was initiated because the 2500 ppm dose group in the first study (M-185485-01-1) was accidentally treated with a 10-fold higher dose for approx. 1 week (Week 22). This caused intercurrent deaths and clinical symptoms of toxicity. Consequently this dose group was terminated in Week 23.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Behaviour and general health condition were observed twice daily (on weekends and public holidays once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Monthly, the animals were examined for neurological disturbances, impairment of dental growth and changes in the eyes and oral mucosa. Moreover, each animal was palpated for skin masses monthly for the first 6 months of treatment and afterwards twice monthly.

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly from the start of treatment (Day 1).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Individually and weekly combined with the body weight examination.
- Compound intake was calculated on the basis of mean weekly relative food consumption and nominal dose levels.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Monthly (see 'Detailed clinical observations')
- How many animals: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 12 months and prior to necropsy.
- Animals fasted: No
- How many animals: All
- Parameters checked: Haematocrit, Haemoglobin, Erythrocyte count, Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Platelet count, Reticulocyte count, Total leucocyte count, and Differential blood count.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- List of organs: Adrenals, aorta, bone marrow (sternum), brain (with medulla oblongata), caecum, cartilage-bone junction, colon, diaphragm, duodenum, epididymides, ethmoid turbinals (head, middle ear), eyes, femur joint, gall bladder, Harderian gland, heart, ileum, jejunum, kidneys, lacrimal gland, larynx, liver, lungs, lymph nodes (mandibular, iliac and mesenteric), mammary gland, muscle (skeletal), nerves (optic and sciatic), oesophagus, oviducts, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (mandibular, sublingual and parotid), seminal vesicles, skin, spinal cord (3 levels), spleen, stomach, testes, thymus, thyroid (with parathyroids), tongue, trachea, urinary bladder, uterus, vagina, and any other tissue showing macroscopic abnormalities.
- Organ weights: Adrenals, brain, heart, kidneys, liver, ovaries with oviducts, spleen, and testes.

HISTOPATHOLOGY: Yes
Histopathology was conducted on organs/tissues indicated above from all animals.

Statistics:

For statistical analysis of in-life findings, haematology and organ weights the 2-tailed significance was evaluated by comparing the treated group with control.

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment-related changes in general health and behaviour, eyes, teeth and oral mucosa as well as during palpation of skin for nodules and masses were not observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

Both sexes of the treated group showed a moderately increased mortality rate (Table 2 under "Any other information on results incl. tables").

Body weight and weight changes:

no effects observed

Description (incidence and severity):

For details see attached data table under "Attached background material".

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Total leukocyte counts showed a marginal statistically significant increase in both sexes of the treated group although there were no significant differences in differential blood counts. The statistical significant increase was considered to be not toxicologically relevant because all values were within the physiological range of control CD-1 mice.

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:

effects observed, non-treatment-related

Description (incidence and severity):

At 2500 ppm, kidney weights were decreased (absolute and relative approx. -11% each) in males to a statistically significant degree. Absolute adrenal weights were as well statistically significantly reduced (approx. -23%) with the relative weights showing a tendency but without significance. However, in the absence of any histopathological correlation in these organs the slight organ weight decreases were considered to be of no toxicological relevance (for details see attached data tables under "Attached background material").

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy a number of macroscopic findings were noted, but they were not associated with any treatment-related histological findings.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males only, centrilobular hepatocellular hypertrophy was observed in 10 animals at 2500 ppm.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Treatment with the test substance did not result in any neoplastic alterations indicative of a carcinogenic potential of the test substance (for details see attached data table under "Attached background material").

Relevance of carcinogenic effects / potential:

No carcinogenic effects found.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other: actual test substance intake: males: 336.85 mg/kg bw/day, females: 407.28 mg/kg bw/day, combined sexes: 372.07 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake: males: 336.85 mg/kg bw/day, females: 407.28 mg/kg bw/day, combined sexes: 372.07 mg/kg bw/day

Key result

Critical effects observed:

no

Table 1: Group mean test material intake (mg/kg bw/day)

Weeks		Dose level (ppm)
		2500
1-78	Males	336.85
	Females	407.28
	Combined sexes	372.07

Table 2: Mortality

Week	Dose level (ppm)
	0		2500
	m	f	m	f
1-26	0	0	0	1
27-52	2	1	3	4
53-78	7	5	12	11
Total deaths	9	6	15	16
% mortality	18	12	30	32

Conclusions:

Overall conclusion (study part A and B)
Dietary administration of the test substance at dose levels up to 2500 ppm to CD-1 mice for 80 weeks was not carcinogenic. This dose level was equivalent to a mean actual substance intake of 337, 407 and 372 mg/kg bw/day in males, females and the combined sexes, respectively.
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 125 ppm, equivalent to 16.6, 19.9 and 18.3 mg/kg bw/day in males, females and the combined sexes, respectively, based on increased mortality rates at both 1250 and 2500 ppm.

Reference 3

Endpoint:

carcinogenicity: oral

Remarks:

combined chronic toxicity and carcinogenicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Nov 1996 - 27 Nov 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Current version adopted in 2018

Deviations:

yes

Remarks:

Thyroid and uterus weights were not measured.

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Guideline in place during study conduct: adopted in 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

Version / remarks:

Adoption not stated

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPP 83-5 (Combined Chronic Toxicity/Oncogenicity Study)

Version / remarks:

Adopted in 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF (Combined Chronic Toxicty/Oncogenicty Study) adopted 1985

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL:CD (IGS) BR

Details on species / strain selection:

The rat was chosen as the test system according to regulatory guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at study initiation: 33 days
- Body weight at receipt (measured for 10% of the animals): male: 55 - 79 g, female: 55 - 84 g
- Fasting periods during study: overnight during urine collection
- Housing: group housed, 5 animals per cage by sex and dose group, in suspended polycarbonate cages with grid floor
- Diet: powdered laboratory rodent diet, Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Ltd., Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: At their respective levels in the diet and water, none of the contaminants known or expected to be present was considered likely to influence the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Nov 1996 To: 13 - 27 Nov 1998

Route of administration:

oral: feed

Vehicle:

other: powdered laboratory rodent diet

Remarks:

Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly by two identical mixes.
- Mixing appropriate amounts: For the highest dose level, the required amount of test material and a small amount of diet were mixed. This mix was then blended with the remaining diet. Subsequent dietary concentrations were prepared by serial dilution.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aliquots of freshly prepared test diets were taken were taken weekly from all concentration. Samples from Weeks 1, 4, 6, 11, 14, 23, 34, 47, 71, 83, 95 and 106 (all concentrations) were analysed. The results were within the range +10% to -15% of nominal.

Duration of treatment / exposure:

52 weeks = 12 months (interim sacrifice)
104 weeks = 24 months (terminal sacrifice)

Frequency of treatment:

Continuously

Dose / conc.:

20 ppm

Remarks:

actual test substance intake: males: 0.8 mg/kg bw/day, females: 1.1 mg/kg bw/day, combined sexes: 1.0 mg/kg bw/day

Dose / conc.:

200 ppm

Remarks:

actual test substance intake: males: 8 mg/kg bw/day, females: 12 mg/kg bw/day, combined sexes: 10.0 mg/kg bw/day

Dose / conc.:

2 000 ppm

Remarks:

actual test substance intake: males: 84 mg/kg bw/day, females: 118 mg/kg bw/day, combined sexes: 101 mg/kg bw/day

Dose / conc.:

4 000 ppm

Remarks:

actual test substance intake: males: 171 mg/kg bw/day, females: 249 mg/kg bw/day, combined sexes: 210 mg/kg bw/day

No. of animals per sex per dose:

20 (interim sacrifice)
50 (terminal sacrifice)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were based on the results of an earlier 90-day dietary rat study (M-141124-01-1).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observation for abnormal behavior, including neuro-muscular coordination and physical appearance were done each morning and as well in the afternoon on Mondays to Fridays (except on holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly prior to weighing.

BODY WEIGHT: Yes
- Time schedule for examinations: 10% of the animals upon receipt. Each animal at randomisation, at the start of treatment, at weekly intervals to Week 14, every second week thereafter and at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- For each sex, the weekly food consumption of each cage of animals was measured at weekly intervals for the first 13 weeks of treatment and approx. every four weeks thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight data: Yes

FOOD EFFICIENCY: Yes
- Body weight gain in % calculated as time-weighted averages from the consumption and body weight gain data

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly for each cage of the numerical last 20 animals from each group over 5-day periods (Monday to Friday) during Weeks 9, 17, 33 and 49.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment in all animals and prior to the interim (Month 12) and terminal kill (Month 24) in the control and high dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 3, 6, 12, 18 and 24 months of treatment.
- Anaesthetic used for blood collection: Yes (ether anaesthesia (Months 3, 6 and 12) or isoflurane anaesthesia (Months 18 and 24))
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Haematocrit (HCT), Haemoglobin (HB), Red blood celIs (RBC), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Platelets (PLT), Prothrombin time (PT), White blood cells (WBC), Neutrophils (NEUT), Lymphocytes (LYMP), Monocytes (MONO), Eosinophils (EOS), Basophils (BASO), Large unstained cells (LUC), Reticulocyte count (RET), and Activated partial thromoplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 3, 6, 12, 18 and 24 months of treatment.
- Anaesthetic used for blood collection: Yes (ether anaesthesia (Months 3, 6 and 12) or isoflurane anaesthesia (Months 18 and 24))
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Total protein (PROT), Albumin (ALB), Total globulin (GLOB), A/G ratio (A/G), Calcium (CA), Phosphate (PO4), Sodium (NA), Potassium (K), Urea (UREA), Creatinine (CREAT), Glucose (GLUC), Total cholesterol (CHOL), Total bilirubin (TBIL), Chloride (CL), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (AP), G-glutamyl transpeptidase (GGT), and Creatine kinase (CPK).

URINALYSIS: Yes
- Time schedule for collection of urine: After 3, 6, 12, 18 and 24 months of treatment.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Appearance (APP), pH (PH), Protein (PROT), Glucose (GLUC), Specific gravity (SG), Colour of Spun deposit (SDEP), Bacteria (BACT), Red blood cells (RBC), Epithelial cells (EPTH), Ketones (KET), Urobilinogen (UBIL), Bilirubin (BIL), Blood (BLD), Phosphate crystals (PO4), Urate crystals (URAT), White blood cells (WBC), Sperm (SPER), Volume (VOL), and Casts (CAST).

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Time schedule: After approx. 52 weeks the first 20 animals/sex/group (interim kill); the remaining animals after 104 weeks (terminal kill)
- Organ weights: Adrenals, brain, heart, kidneys, liver, ovaries, spleen, and testes with epididymides.
- List of organs: Adrenals, aorta (abdominal), bile duct*, brain, caecum, colon, diaphragm, duodenum, epididymides, eyes, femur and joint, Harderian gland, head, heart, ileum, jejunum, kidneys, lacrimal gland (exorbital), liver, lungs (inflated), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), nerves (optic and sciatic), oesophagus, oviducts, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary, sublingual and parotid), seminal vesicles, skin, spinal cord (3 levels), spleen, sternum, stomach, testes, thymus, thyroid (with parathyroids), tongue, trachea, urinary bladder, uterus, vagina, and any other tissue showing macroscopic abnormalities.

* Taken from animals killed from Week 62 of treatment onwards.

HISTOPATHOLOGY: Yes
Histopathology was conducted on organs/tissues indicated above from all animals.

Other examinations:

None.

Statistics:

The significance of differences between control and treated groups was analysed by either parametric or non-parametric statistical tests as appropriate. A maximum 2-tailed probability value of 5% (p <0.05) was considered statistically significant.
The following convention has been used to indicate statistical significance:
* p <0.05
** p <0.01
*** p <0.001

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm a slight increased incidence of urogenital staining for males from approx. Week 41 onwards (non-adverse).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no treatment-related effect on mortality during the course of the study.
The incidence of mortality after 12 months of treatment was 2/70, 1/70, 3/70, 1/70 and1/70 for males and 3/70, 5/70, 1/70, 2/70 and 5/70 for females, at 0, 20, 200, 2000 and 4000 ppm, respectively.
The incidence of mortality at 24 months was 23/50, 27/50, 24/50, 19/50 and 17/50 for males and 33/50, 28/50, 27/50, 34/50 and 31/50 for females, at 0, 20, 200, 2000 and 4000 ppm, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm, body weight gain of females was reduced by 8% (non-adverse). This was mainly due to a marked reduction during Week 1 (38%) although a trend was seen throughout the study. Males of the same group showed as well a reduction in Week 1 (19%) but body weight gain in general was unaffected (see Table 1 under "Any other information on results incl. tables").

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

At 4000 ppm food intakes of both sexes were reduced during Week 1 but reached the control level thereafter (see Table 2 and 3 under "Any other information on results incl. tables").

Food efficiency:

no effects observed

Description (incidence and severity):

At 4000 ppm food conversion efficiency of females were reduced by 31% during Week 1 but reached the control level thereafter.

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

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm a slight (18 - 27%) but consistent increase in water consumption was seen throughout the study for females (see Table 4 under "Any other information on results incl. tables").

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Single parameters of treated animals distributed through all dose levels were statistically significantly changed compared to control. However, all changes were without any dose-response dependence, therefore, they are of no toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly increased urea and creatinine levels were observed at several time points at 2000 and 4000 ppm in both sexes (for details see attached result tables under "Attached background material"). However, all values were within the physiological range of this rat strain.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At Month 3, 6 and 12 an increased incidence of ketones present in the urine was seen in males treated at 2000 and 4000 ppm and also at Month 18 in males given 4000 ppm (see Table 5 under "Any other information on results incl. tables").

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At interim kill, relative liver weights were slightly increased for males (16%) and females (14%) treated at 4000 ppm. At 2000 ppm, relative liver weights were increased (8%) for males; however, this is considered not toxicologically relevant as there was no histopathological correlate at terminal kill.
At terminal kill, both absolute and relative kidney weights were decreased for males at 4000 ppm (12% and 9%, respectively) and at 2000 ppm (10% and 13%, respectively). Furthermore, relative liver weights were increased (8%) for males given 4000 ppm (see Table 6 under "Any other information on results incl. tables").

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At interim and terminal kill, an increased incidence of centrilobular hepatocytic hypertrophy (minimal to slight) was seen in males receiving 4000 ppm.
At terminal kill, an increased incidence and severity of progressive nephropathy was observed in females treated at 4000 ppm (see Table 7 under "Any other information on results incl. tables").

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Administration of the test substance did not result in any alterations to indicate a carcinogenic potential from animals killed after 52 or 104 weeks of treatment (for details see attached data tables under "Attached background material").

Other effects:

no effects observed

Relevance of carcinogenic effects / potential:

No carcinogenic effects found.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake: males: 171 mg/kg bw/day, females: 249 mg/kg bw/day, combined sexes: 210 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: actual test substance intake: males: 8 mg/kg bw/day, females: 12 mg/kg bw/day, combined sexes: 10 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: actual test substance intake: males: 84 mg/kg bw/day, females: 118 mg/kg bw/day, combined sexes: 101 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

other: urinary and hepatobiliary

Organ:

kidney

liver

other: increased ketone content in urine in males, decreased absolute and relative kidney weights in males; increased relative liver weights and hepatocytic hypertrophy in males at 4000 ppm

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Group mean body weights (g)

Weeks	Dose level (ppm)
	Males					Females
	0	20	200	2000	4000	0	20	200	2000	4000
0-14	347 (−)	338 (97)	338 (97)	328 (95)	316 (91)	151 (−)	151 (100)	150 (99)	146 (97)	132 (87)
14-28	108 (−)	110 (102)	107 (99)	108 (100)	107 (99)	39 (−)	45 (115)	43 (110)	37 (95)	37 (95)
28-52	99 (−)	107 (108)	99 (100)	103 (104)	88 (89)	70 (−)	80 (114)	81 (116)	72 (103)	60 (86)
52-78	69 (−)	76 (110)	60 (87)	62 (90)	61 (88)	81 (−)	85 (105)	64 (79)	66 (81)	45 (56)
0-104	606 (−)	650 (107)	618 (102)	635 (105)	593 (98)	359 (−)	385 (107)	365 (102)	345 (96)	332 (92)

() Percentage of control values

 

Table 2: Group mean food consumption (g/animal/day)

Weeks	Dose level (ppm)
	Males					Females
	0	20	200	2000	4000	0	20	200	2000	4000
1-12	27 (−)	27 (100)	27 (100)	27 (100)	26 (96)	21 (−)	21 (100)	21 (100)	21 (100)	20 (95)
13-26	26 (−)	27 (104)	26 (100)	26 (100)	26 (100)	20 (−)	22 (110)	21 (105)	22 (110)	21 (105)
30-51	27 (−)	27 (100)	27 (100)	27 (100)	27 (100)	22 (−)	23 (105)	22 (100)	23 (105)	22 (100)
54-74	27 (−)	27 (100)	27 (100)	27 (100)	26 (96)	22 (−)	24 (109)	23 (105)	23 (105)	23 (105)
79-103	26 (−)	27 (104)	26 (100)	27 (104)	27 (104)	23 (−)	24 (104)	24 (104)	24 (104)	24 (104)
1-103	27 (−)	27 (100)	27 (100)	27 (100)	26 (96)	22 (−)	22 (100)	22 (100)	22 (100)	22 (100)

() Percentage of control values

 

Table 3: Group mean test substance intake (mg/kg bw/day)

Weeks		Dose level (ppm)
		20	200	2000	4000
1-104	Males	0.8	8	84	171
	Females	1.1	12	118	249
	Combined sexes	1.0	10	101	210

 

Table 4: Group mean water consumption (g/animal/day)

Week	Dose level (ppm)
	Males					Females
	0	20	200	2000	4000	0	20	200	2000	4000
9	35 (−)	32 (91)	32 (91)	34 (97)	35 (100)	28 (−)	28 (100)	30 (107)	31 (111)	33 (118)
17	31 (−)	31 (100)	29 (94)	32 (103)	33 (106)	27 (−)	29 (107)	29 (107)	28 (104)	33 (122)
33	29 (−)	30 (103)	28 (97)	31 (107)	32 (110)	34 (−)	31 (91)	34 (100)	30 (88)	41 (121)
49	28 (−)	32 (114)	31 (111)	29 (104)	33 (118)	33 (−)	35 (106)	38 (115)	31 (94)	42 (127)

() Percentage of control values

 

Table 5: Group incidence of ketones present in urine

Month	Dose level (ppm)
	Males					Females
	0	20	200	2000	4000	0	20	200	2000	4000
3	1/10	0/10	3/10	9/10	10/10	2/10	0/10	0/10	0/10	2/10
6	0/10	0/9	2/10	9/10	10/10	3/10	0/10	1/10	1/10	2/10
12	0/10	1/9	0/10	5/10	9/10	0/10	0/10	0/10	0/10	0/10
18	0/10	0/10	0/10	2/10	5/10	0/10	0/10	0/10	0/10	0/10
24	0/10	0/10	0/10	2/10	2/10	1/10	0/10	1/9	0/10	0/10

 

Table 6: Treatment-related effects on organ weights at interim and terminal kill

	Dose level (ppm)
	Males					Females
	0	20	200	2000	4000	0	20	200	2000	4000
Interim kill
Relative liver weight (%)	2.67 (−)	2.76 (103)	2.68 (100)	2.88 (108)	3.10 (116)	2.78 (−)	2.97 (107)	2.77 (100)	2.95 (106)	3.16 (114)
Terminal kill
Absolute kidney weights (g)	4.11 (−)	4.18 (102)	4.29 (104)	3.68 (90)	3.60 (88)	No treatment-related effects
Relative kidney weight (%)	0.53 (−)	0.51 (96)	0.56 (106)	0.46 (87)	0.48 (91)
Relative liver weight (%)	2.56 (−)	2.54 (99)	2.70 (105)	2.53 (99)	2.77 (108)

() Percentage of control values

Table 7: Histopathological treatment-related effects at interim and terminal kill

	Dose level (ppm)
	Males					Females
	0	20	200	2000	4000	0	20	200	2000	4000
Interim kill
Liver
Centrilobular hepatocytic hypertrophy	1/19	2/20	0/19	5/20	15/20	No treatment-related effects
Terminal kill
Liver
Centrilobular hepatocytic hypertrophy	0/26	0/22	0/25	0/31	12/33	No treatment-related effects
Kidney
Progressive nephropathy	No treatment-related effects					25/50	22/50	23/50	17/50	31/50
Minimal						16	13	16	12	11
Slight						8	7	4	5	10
Moderate						0	2	2	0	6
Sever						1	0	1	0	4

Conclusions:

Under the conditions of the present study, after chronic oral exposure to the test substance, a LOAEL of 101 mg/kg bw/day for combined sexes was identified based on urinary/renal findings. Hence, no classification for specific target organ toxicity is required according to Regulation (EC) No. 1272/2008.
No effects indicating a carcinogenic potential of the test substance were observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

210 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Two oral carcinogenicity studies (RL1) conducted according to OECD TG 451 (mice, two parts) or OECD TG 453 (rat) and in compliance with GLP are available.

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

The available data on carcinogenicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.

Additional information

The potential of the test substance to cause carcinogenic effects was examined in rats and mice. Two oral carcinogenicity studies (RL1) conducted according to OECD TG 451 (mice, two parts (A and B)) or OECD TG 453 (rat) and in compliance with GLP are available.

 

In a combined chronic toxicity/carcinogenicity study (M-191414-01-1, 1999), 70 rats/sex/dose received the test substance via the diet at dose levels of 20, 200, 2000 and 4000 ppm (corresponding to 1.0, 10, 101 and 210 mg/kg bw/day actual test substance intake for combined sexes) for 104 weeks (2 years). After 52 weeks, 20 animals of each group were subjected to an interim necropsy. Organ weights of these animals were recorded and tissues microscopically examined. The remaining animals continued treatment until termination of the study after 104 weeks. All animals were observed daily for clinical signs and palpated weekly for tissue masses. Body weight was recorded at the start of treatment, at weekly intervals to Week 14, and every second week thereafter until necropsy. Food consumption was monitored at weekly intervals for the first 13 weeks of treatment and approx. every 4 weeks thereafter. Water consumption was measured during Month 3, 5, 9 and 12. Haematology, clinical chemistry and urinalysis were carried out at 3, 6, 12, 18 and 24 months of treatment. Ophthalmoscopy was performed prior to treatment and at 12 and 24 months of treatment. At terminal necropsy, the weights of selected organs were recorded and a full list of tissues from all animals was examined histopathologically.

At 20 and 200 ppm, no treatment-related effects were observed. At 2000 ppm, an increased incidence in the presence of ketones in the urine of male animals was observed after 3, 6 and 12 months of treatment. Absolute and relative kidney weights were decreased in males at terminal sacrifice (10 and 13%, respectively). At interim kill only, relative liver weights were slightly increased (8%) in males, however, without a histopathological correlate at terminal kill. Therefore, this finding was considered adaptive and not toxicologically relevant at this dose level.

At 4000 ppm, palatability of the diet affected body weight gain, food consumption and food efficiency during the first week of treatment in both sexes. All parameters recovered to a great extend during the study compared with control. An increased incidence in the presence of ketones in the urine of male animals was observed after 3, 6 and 12 months of treatment. At interim kill, relative liver weights were slightly increased (16 and 14% for males and females, respectively) in both sexes compared to control. Increased relative liver weights (8%) were also observed for male animals at terminal kill. Absolute and relative kidney weights were decreased (12 and 9%, respectively) for males at terminal kill. An increased incidence of centrilobular hepatocytic hypertrophy in the liver of male animals was observed at both examination time points (after 12 and 24 months of treatment). An increase in the incidence and severity of progressive nephropathy in the kidneys was observed after 24 months of treatment in female animals.

In summary, the examinations revealed an increased incidence of ketones in the urine as well as decreased absolute and relative kidney weights in male animals starting at 2000 ppm. Hence, the NO(A)EL for systemic toxicity was set at 200 ppm, corresponding to an actual test substance intake of 8 mg/kg bw/day for male animals, 12 mg/kg bw/day for female animals and 10 mg/kg bw/day for combined sexes (M-191414-01-1, 1999).

Administration of the test substance did not result in any alterations indicating a carcinogenic potential in animals treated with the test substance for 52 or 104 weeks. No neoplastic/carcinogenic effects were found up to the highest dose level. Therefore, the NOAEL for carcinogenic effects was set at 4000 ppm, equivalent to 171 mg/kg bw/day in males, 249 mg/kg bw/day in females and 210 mg/kg bw/day for combined sexes (M-191414-01-1).

 

The second carcinogenicity study, performed in CD-1 mice, was divided into two parts (A and B) due to an accidental overdosing (10-fold higher) in the high dose group during the first study part (A) (M-185485-01-1, M-185504-01-1). Therefore, the second study part (B) was only conducted with a concurrent control group and the highest dose level of 2500 ppm. In the following both study parts (A and B) are summarised. Oral exposure of 50 mice/sex/dose to the test substance was achieved via dietary treatment at dose levels of 12.5, 125, 1250 and 2500 ppm (corresponding to 1.88, 18.24, 193 and 372 mg/kg bw/day for combined sexes) for 18 months.

Animals were observed daily for mortality and clinical signs of toxicity. They were palpated for masses monthly for the first 6 months and twice monthly thereafter till study termination. Body weights and food consumption were recorded weekly. Haematological investigations were conducted after 12 months and just prior to study termination. All animals including decedents were examined macroscopically, both externally and internally, and a full range of tissues preserved and processed to slides. Tissues from all animals were histopathologically examined using light microscopy.

At 2500 ppm, mortality was statistically significantly higher in both sexes compared to the pooled controls of both studies. In males at 1250 ppm, mortality was also slightly increased. Behaviour and general health, the incidence of palpable masses, body weight gain, food consumption and organ weights were not affected by treatment up to 2500 ppm, the highest dose level tested. There were no treatment-related effects on haematological parameters. Statistically significantly higher total leukocyte counts were observed at 125 ppm and above when compared to concurrent controls. Because there was no clear dose-response relationship, no histopathological correlate, and all values were within the background control data range, this observation was regarded as not toxicologically relevant.

At 2500 ppm, increased centrilobular hepatocyte hypertrophy in male animals was observed. There were no other treatment-related findings recorded. No evidence of a carcinogenic potential of the test substance was observed at any dose level.

Based on the increased mortality rate, the NOAEL for systemic toxicity was set at 125 ppm, equating to an actual test substance intake of 16.6, 19.9 and 18.3 mg/kg bw/day in males, females and combined sexes, respectively.

Because no neoplastic/carcinogenic effects were observed, the NOAEL for carcinogenicity was set at 2500 ppm, equating to an actual test substance intake of 337, 407 and 372 mg/kg bw/day in males, females and combined sexes, respectively.