(3R)-3,7-dimethyloct-6-enenitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2007 - 12 October 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

20 March 2007 - 12 October 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The structures of the target and source substances are identical and differ only with respect to the ratio of enantiomers where the target substance is a single pure L-isomer and the source substance is an equimolar mixture of L and D isomers.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellyl nitrile, is a mono-constituent substance (EC No. 695-909-8, CAS no. 35931-93-2).
The source substance, DL-Citronellyl nitrile, is a mono-constituent substance (EC No. 257-288-8, CAS no. 51566-62-2).
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the only difference between target and source molecules is the enantiomeric ratio. In a non-chiral environment the target and source chemicals will have identical properties but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). Therefore, as a precaution for the developmental toxicity endpoint it is suggested that the NOAEL 250 mg/kg bw/day for L-Citronellyl nitrile is used instead of 500 mg/kg bw/day, as it is not known which form is more potent in vivo. All other endpoints are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: male: five to eight weeks old.
- Weight at study initiation: (P) Males: 147 to 183g; Females: 13 1 to 162g
- Housing: polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Acclimatation: eight days
- Temperature: 21 ± 2°C
- Humidity: 55 ± 15%
- Air changes: at least fifteen air changes per hour
- Photoperiod: twelve hours continuous light and twelve hours darkness
- Enrichment: wooden chew blocks and cardboard fun tunnels

Route of administration:

oral: gavage

Details on route of administration:

By a stainless steel cannula attached to a disposable plastic syringe.

Vehicle:

corn oil

Duration of treatment / exposure:

The test material was administered for ninety consecutive days.

Frequency of treatment:

daily

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Observations and examinations performed and frequency:

Clinical Observations:
signs of toxicity, ill-health or behavioural change immediately before and after dosing and one and five hours after dosing during the working week.

Functional Observations:
Signs of functional/behavioural toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Functional Performance Tests:
Motor Activity: The evaluation period was thirty minutes for each animal.
ForelimbBindlimb Grip Strength.
Sensory Reactivity: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Startle reflex, Blink reflex

Behavioural parameters:
Gait, Tremors, Twitches, Convulsions, Salivation, Pilo-erection, Exophthalmia, Lachrymation, HyperMypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation.

Bodyweight:
Recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.

Food Consumption:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption:
Water intake was observed daily.

Ophthalmoscopic Examination:
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12).

Haematological, blood chemical and urinalysis investigations were performed on all animals from each test and control group during Week 7 and at the end of the study (Day 90).

Oestrous Cycles:
During Weeks 6 and 7 and Weeks 12 and 13 a vaginal smear was taken daily.

Pathology:
On completion of the dosing period.

Organ Weights:
Adrenals, Brain, Epididyrnides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus.

Histopathology

Semen Assessment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinically observable signs of toxicity were detected in test or control animals throughout the study period.
Incidental findings of increased salivation, hunched posture and tiptoe gait, were evident in 300 mgikg/day animals throughout the treatment period. Incidents of increased salivation were also evident in 100, 30 and 10 (males only) mg/kg/day animals. Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test material formulation and considered not to be an indication of systemic toxicity. Isolated instances of generalised fur loss, wet fur, wounds, scab formation or redbrown stained snout were evident in a number of control and treated animals throughout the dosing period. Such observations are commonly observed in laboratory maintained rats and in view of the sporadic nature of these findings were considered to be entirely incidental and unrelated to treatment. Control females showed episodes of noisy respiration whilst one control male developed swollen limbs between Days 41 and 60 and an abnormal gait between days 41 and 49. These were isolated incidental
findings and unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No adverse effect on bodyweight development was detected. Bodyweight gain in test animals during the treatment period was similar to that of controls.
Females treated with 10 mgkglday showed a statistically significant increase in bodyweight gain during Week 3 whilst females treated with 300, 100 and 30 mgkglday showed a statistically significant reductions in bodyweight gain during Week 8. In the absence of a dose-related response the intergroup differences were considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption during the study period. Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory maintained rats of this age and strain.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically significant changes in the haematological parameters measured.
During the Week 7 assessments, males from all treated groups showed a statistically significant reduction in haemoglobin. Males treated with 300 and 100 mg/kg/day also showed a reduction in mean cell volume and neutrophils. In the absence of a dose-related response these intergroup differences were considered of no toxicological importance. Males treated with 300 and 100 mg/kg/day showed a statistically significant reduction in mean cell haemoglobin and an increase in lymphocyte count (300 mg/kg/day only). Reductions in mean cell haemoglobin were still evident during Week 13 assessments together with statistically significant reductions in mean cell haemoglobin concentration for 300 mgikglday males, haematocrit levels for 100 mglkglday males and an increase in erythrocyte count for 300 mgikglday. In the absence of any supporting data in the bone marrow to suggest an effect of treatment the intergroup differences were considered of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

During the Week 7 assessments, a statistically significant increase in total protein was evident for females treated with 300 mgkglday. Increases in total protein were still evident during Week 13 assessments for females treated with 300 mgikglday. Males from this treatment group also showed a statistically significant increase in albumin levels.
No such effects were detected in animals of either sex treated with 100,30 or 10 mgikglday. During Week 7 assessments males from all treated groups showed a statistically significant reduction in alanine aminotransferase. In the absence of a dose-related response or supporting data to suggest an effect of treatment these intergroup differences were considered of no toxicological importance. Females treated with 300 mgikglday showed a statistically significant reduction in albumin/globulin ratio during Week 7 assessments. This intergroup difference was considered to be the result of higher than normal control values and was of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the urinalytical parameters measured.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 300 mgkglday showed a statistically significant increase in liver weight both absolute and relative to terminal bodyweight. The effect on liver weight also extended to males treated with 100 mgkglday. No such effects were detected in females treated with 100 mgkgiday or in animals of either sex treated with 30 or 10 mg1kg/day. Males treated with 100 and 30 mgkglday showed a statistically significant increase in heart weight both absolute and relative to terminal bodyweight. Females treated with 300 mgkglday showed a statistically significant increase in adrenal and kidney weight both absolute and relative to terminal bodyweight. The majority of individual values were within the normal range for rats of the strain and age used and in the absence of any histological correlates the intergroup differences were considered not to be of any toxicologically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically significant macroscopic abnormalities were detected.
Hydronephrosis was evident in one male treated with 300 mgkglday and in two males and one female treated with 30 rngkglday. One male treated with 30 mgkglday also showed small and flaccid testes whilst two females treated with 300 mgkglday showed in the ovaries, a fluid fill sac. Finally, lung changes (dark, pale or red lobes) were evident in one male and one female treated with 10 or 100 rngkglday. In the absence of a dose-related response or any histological correlates the intergroup differences were considered to be of no toxicological importance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

LIVER: Marginal centrilobular hepatocyte enlargement was observed among animals of either sex treated with 300 mgkglday 0, <0.05). Two instances of centrilobular hepatocyte enlargement were seen among males and one female treated with 100 mgkglday. It is possible that the effect of treatment extended to animals at this dose level, although hepatocyte enlargement is occasionally observed as a spontaneous change among untreated control rats. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.
BONE MARROW: A higher incidence of lower grades of severity of adipose infiltration of the marrow, indicative of increased marrow cellularity, was seen for females only treated with 300 mgkglday but not at any other treatment level. This was considered to be a marginal effect that did not attain statistical significance.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and was of no toxicological importance.
There were no toxicologically significant changes in the functional performance parameters measured. In the absence of a dose-related response or any supporting clinical observations to suggest an effect of neurotoxicity, some findings were considered to be of no toxicological significance. Females treated with 300 and 100 mglkglday showed a statistically significant increase in mean hind limb grip strength. This effect was confined to one out of the three tests for this parameter and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, this finding was considered to be of no toxicological significance.
There were no treatment-related effects detected in sperm motility values, morphological assessments or in homogenisation-resistant spermatid counts.

Details on results:

Remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.
Increased hepatic activity of this type and in the absence of associated inflammatory or degenerative changes is considered a normal adaptive biological response to xenobiotics and is usually the result of detoxification mechanisms involving hepatic enzyme induction.
The remaining histopathological changes were evident in the bone marrow. A higher incidence of lower grades of severity of adipose infiltration of the marrow, indicative of increased marrow cellularity, was seen for females only treated with 300 mgkglday. These changes may possibly suggest the initiation of a slight anaemic response to the test material, however haematological determination revealed no significant changes and the effects were considered to be marginal and did not attain statistical significance.
No such effects were detected in animals of either sex treated with 30 or 10 mgkglday.

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood vessel

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood vessel

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Conclusions:

The oral administration of the test substance oo rats for a period of ninety consecutive days at dose levels of 10, 30, 100 and 300 mgkglday resulted in treatment-related effects in animals of either sex treated with 300 and 100 mgkglday. No such changes were demonstrated at 30 or 10 mgkglday. The 'No Observed Effect Level' (NOEL) was, therefore considered to be 30 mg/kg!day. 300 mgkglday may be regarded as a "No Observed Adverse Effect Level" (NOAEL).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethyloct-6-enenitrile

EC Number:

257-288-8

EC Name:

3,7-dimethyloct-6-enenitrile

Cas Number:

51566-62-2

Molecular formula:

C10H17N

IUPAC Name:

3,7-dimethyloct-6-enenitrile

Test material form:

liquid

Details on test material:

Description: clear colourless liquid
Storage conditions: room temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: male: five to eight weeks old.
- Weight at study initiation: (P) Males: 147 to 183g; Females: 13 1 to 162g
- Housing: polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Acclimatation: eight days
- Temperature: 21 ± 2°C
- Humidity: 55 ± 15%
- Air changes: at least fifteen air changes per hour
- Photoperiod: twelve hours continuous light and twelve hours darkness
- Enrichment: wooden chew blocks and cardboard fun tunnels

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

By a stainless steel cannula attached to a disposable plastic syringe.

Vehicle:

corn oil

Duration of treatment / exposure:

The test material was administered for ninety consecutive days.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Examinations

Observations and examinations performed and frequency:

Clinical Observations:
signs of toxicity, ill-health or behavioural change immediately before and after dosing and one and five hours after dosing during the working week.

Functional Observations:
Signs of functional/behavioural toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Functional Performance Tests:
Motor Activity: The evaluation period was thirty minutes for each animal.
ForelimbBindlimb Grip Strength.
Sensory Reactivity: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Startle reflex, Blink reflex

Behavioural parameters:
Gait, Tremors, Twitches, Convulsions, Salivation, Pilo-erection, Exophthalmia, Lachrymation, HyperMypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation.

Bodyweight:
Recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.

Food Consumption:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption:
Water intake was observed daily.

Ophthalmoscopic Examination:
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12).

Haematological, blood chemical and urinalysis investigations were performed on all animals from each test and control group during Week 7 and at the end of the study (Day 90).

Oestrous Cycles:
During Weeks 6 and 7 and Weeks 12 and 13 a vaginal smear was taken daily.

Pathology:
On completion of the dosing period.

Organ Weights:
Adrenals, Brain, Epididyrnides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus.

Histopathology

Semen Assessment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinically observable signs of toxicity were detected in test or control animals throughout the study period.
Incidental findings of increased salivation, hunched posture and tiptoe gait, were evident in 300 mgikg/day animals throughout the treatment period. Incidents of increased salivation were also evident in 100, 30 and 10 (males only) mg/kg/day animals. Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test material formulation and considered not to be an indication of systemic toxicity. Isolated instances of generalised fur loss, wet fur, wounds, scab formation or redbrown stained snout were evident in a number of control and treated animals throughout the dosing period. Such observations are commonly observed in laboratory maintained rats and in view of the sporadic nature of these findings were considered to be entirely incidental and unrelated to treatment. Control females showed episodes of noisy respiration whilst one control male developed swollen limbs between Days 41 and 60 and an abnormal gait between days 41 and 49. These were isolated incidental
findings and unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No adverse effect on bodyweight development was detected. Bodyweight gain in test animals during the treatment period was similar to that of controls.
Females treated with 10 mgkglday showed a statistically significant increase in bodyweight gain during Week 3 whilst females treated with 300, 100 and 30 mgkglday showed a statistically significant reductions in bodyweight gain during Week 8. In the absence of a dose-related response the intergroup differences were considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption during the study period. Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory maintained rats of this age and strain.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically significant changes in the haematological parameters measured.
During the Week 7 assessments, males from all treated groups showed a statistically significant reduction in haemoglobin. Males treated with 300 and 100 mg/kg/day also showed a reduction in mean cell volume and neutrophils. In the absence of a dose-related response these intergroup differences were considered of no toxicological importance. Males treated with 300 and 100 mg/kg/day showed a statistically significant reduction in mean cell haemoglobin and an increase in lymphocyte count (300 mg/kg/day only). Reductions in mean cell haemoglobin were still evident during Week 13 assessments together with statistically significant reductions in mean cell haemoglobin concentration for 300 mgikglday males, haematocrit levels for 100 mglkglday males and an increase in erythrocyte count for 300 mgikglday. In the absence of any supporting data in the bone marrow to suggest an effect of treatment the intergroup differences were considered of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

During the Week 7 assessments, a statistically significant increase in total protein was evident for females treated with 300 mgkglday. Increases in total protein were still evident during Week 13 assessments for females treated with 300 mgikglday. Males from this treatment group also showed a statistically significant increase in albumin levels.
No such effects were detected in animals of either sex treated with 100,30 or 10 mgikglday. During Week 7 assessments males from all treated groups showed a statistically significant reduction in alanine aminotransferase. In the absence of a dose-related response or supporting data to suggest an effect of treatment these intergroup differences were considered of no toxicological importance. Females treated with 300 mgikglday showed a statistically significant reduction in albumin/globulin ratio during Week 7 assessments. This intergroup difference was considered to be the result of higher than normal control values and was of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the urinalytical parameters measured.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 300 mgkglday showed a statistically significant increase in liver weight both absolute and relative to terminal bodyweight. The effect on liver weight also extended to males treated with 100 mgkglday. No such effects were detected in females treated with 100 mgkgiday or in animals of either sex treated with 30 or 10 mg1kg/day. Males treated with 100 and 30 mgkglday showed a statistically significant increase in heart weight both absolute and relative to terminal bodyweight. Females treated with 300 mgkglday showed a statistically significant increase in adrenal and kidney weight both absolute and relative to terminal bodyweight. The majority of individual values were within the normal range for rats of the strain and age used and in the absence of any histological correlates the intergroup differences were considered not to be of any toxicologically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically significant macroscopic abnormalities were detected.
Hydronephrosis was evident in one male treated with 300 mgkglday and in two males and one female treated with 30 rngkglday. One male treated with 30 mgkglday also showed small and flaccid testes whilst two females treated with 300 mgkglday showed in the ovaries, a fluid fill sac. Finally, lung changes (dark, pale or red lobes) were evident in one male and one female treated with 10 or 100 rngkglday. In the absence of a dose-related response or any histological correlates the intergroup differences were considered to be of no toxicological importance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

LIVER: Marginal centrilobular hepatocyte enlargement was observed among animals of either sex treated with 300 mgkglday 0, <0.05). Two instances of centrilobular hepatocyte enlargement were seen among males and one female treated with 100 mgkglday. It is possible that the effect of treatment extended to animals at this dose level, although hepatocyte enlargement is occasionally observed as a spontaneous change among untreated control rats. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.
BONE MARROW: A higher incidence of lower grades of severity of adipose infiltration of the marrow, indicative of increased marrow cellularity, was seen for females only treated with 300 mgkglday but not at any other treatment level. This was considered to be a marginal effect that did not attain statistical significance.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and was of no toxicological importance.
There were no toxicologically significant changes in the functional performance parameters measured. In the absence of a dose-related response or any supporting clinical observations to suggest an effect of neurotoxicity, some findings were considered to be of no toxicological significance. Females treated with 300 and 100 mglkglday showed a statistically significant increase in mean hind limb grip strength. This effect was confined to one out of the three tests for this parameter and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, this finding was considered to be of no toxicological significance.
There were no treatment-related effects detected in sperm motility values, morphological assessments or in homogenisation-resistant spermatid counts.

Details on results:

Remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.
Increased hepatic activity of this type and in the absence of associated inflammatory or degenerative changes is considered a normal adaptive biological response to xenobiotics and is usually the result of detoxification mechanisms involving hepatic enzyme induction.
The remaining histopathological changes were evident in the bone marrow. A higher incidence of lower grades of severity of adipose infiltration of the marrow, indicative of increased marrow cellularity, was seen for females only treated with 300 mgkglday. These changes may possibly suggest the initiation of a slight anaemic response to the test material, however haematological determination revealed no significant changes and the effects were considered to be marginal and did not attain statistical significance.
No such effects were detected in animals of either sex treated with 30 or 10 mgkglday.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood vessel

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood vessel

3

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Applicant's summary and conclusion

Conclusions:

The oral administration of the test substance oo rats for a period of ninety consecutive days at dose levels of 10, 30, 100 and 300 mgkglday resulted in treatment-related effects in animals of either sex treated with 300 and 100 mgkglday. No such changes were demonstrated at 30 or 10 mgkglday. The 'No Observed Effect Level' (NOEL) was, therefore considered to be 30 mg/kg!day. 300 mgkglday may be regarded as a "No Observed Adverse Effect Level" (NOAEL).