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

Endpoint summary

Description of key information

This endpoint was fulfilled using read across from 3,7-dimethyloct-6-enenitrile (EC 257-288-8 / CAS 51566-62-2), for which the following results were obtained.

90 -Day Repeated Dose Study:

Repeated dose toxicity was assessed according to test guideline OECD 408. The oral administration of the test substance to 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 mg/kg/day. 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).

One-generation reproduction toxicity study:

A One-generation reproduction toxicity study, based on OECD test guideline 415 was also used to assess repeated dose toxicity. Based on the results of this study, where animals were treated at doses 75, 200 and 500 mg/kg/day, the no-observable-adverse-effect-level (NOAEL) for toxicity of the test substance is 200 mg/kg/day. Increased incidences of excess salivation and/or ungroomed coat occurred in P generation male and/or female rats in the 500 mg/kg/day dosage group. However, these clinical signs were not considered an adverse effect of the test item. At 500 mg/kg/day, sporadic reductions in body weight gains occurred in male rats prior to cohabitation, followed by persistent reductions in weight gain through the end of the study. Non-reproductive organ weights were affected in both sexes at the end of the dosage period, as well as terminal body weights. Increased brain and spleen weights occurred in male rats treated with 500 mg/kg/day test item, while increased liver and kidney weights occurred in both sexes in this same dosage group. The toxicological significance of the increased organ weights was unable to be determined because there were no microscopic findings that could be correlated with the changes in organ weights.

The OECD 415 was not tested at 300 mg/kg and because 500 mg/kg was found to have an adverse effect, by default 200 mg/kg became the NOAEL (because it is the next highest dose level). The substance was not tested at 300 mg/kg in the OECD 415 study, and so it cannot refute the findings of the 90 day study. Therefore the overall NOAEL is considered to be 300 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same study

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).

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Oral (Gavage) One-Generation Reproduction Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 FEB 2009 - 01 FEB 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD guidelines for the testing of chemicals. One-generation reproduction toxicity, No. 415 section 4 Health Effects (Pink pages); adopted May 26, 1983. Organisation for Economic Co-operation and Development.

Version / remarks:

Deviations see Deviation (other information):
-Acclimation
-Formulation Preparation
-Dosage Administration
-Clinical Observations
-Litter Observations
-Sexual Maturation
-Gross Necropsy, Organ Weights and Tissue Retention

Deviations:

yes

Remarks:

The deviations from the protocol had no impact on the study outcome or interpretation of the data because, relative to the total number of animals evaluated and/or the number of data points collected per parameter, the deviations were not significant.

Principles of method if other than guideline:

The requirements of the Organisation for Economic Co-operation and Development Guidelines for Testing of Chemicals No. 415 were used as the basis for study design.

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: 37 days (age at arrival), female 65 days (age at arrival)
- Weight at study initiation: (P) Males: 129 - 160 g; Females: 224 - 266 g
- Housing: individually housed in stainless steel, wire-bottomed cages; during cohabitation, each pair of male and female rats was housed in the male rat's cage; P generation female rats were individually housed in nesting boxes beginning no later than day of (presumed) gestation 20
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 19°C to 25°C
- Humidity: 30% to 70%
- Air changes: a minimum of ten changes per hour
- Photoperiod: 12-hours light:12-hours dark fluorescent light cycle
- Enrichment: supplied to all rats during the course of the study

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Results indicate that the formulations are within the acceptable limits of ±15% of nominal concentrations. The formulations are also within the acceptable limits of ≤5% RSD for homogeneity

DIET PREPARATION
Dosages were adjusted weekly for body weight changes and given at approximately the same time each day. Any dams in the process of parturition were not given the test substance and/or vehicle until the following work day. No dam missed more than one daily dosage administration, and such events were noted in the raw data. Prepared formulations were stirred continuously during dosage administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 10, 30, 100, or 300 mg/kg/day of test item
- Amount of vehicle: 4 mL/kg for 0 mg/kg/day of test item

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose formulations were analyzed by HPLC-UV.
The method was validated for the analysis of dose formulations at concentrations ranging from 1.2 mg/mL to 150 mg/mL of the test item in corn oil.
Results for all dose formulations met the acceptance criteria for concentration (within ±15% of nominal concentration) and homogeneity (≤5% relative standard deviation) except for end of study samples for Group II (18.75 mg/mL), Group III (50 mg/mL), Group IV (125 mg/mL) are out of specification (+27.3%, +20.3% and +20.0%, respectively). Time zero stability samples at 125 mg/mL were not within the acceptable limits of ±15% of nominal concentration, (+25.6%); however, the result was accepted and used as the reference concentration for formulation stability evaluations.

Duration of treatment / exposure:

male: once daily beginning 83 days before the cohabitation period, through the cohabitation period (maximum of 13 days) and continuing until the day before euthanasia
female: once daily beginning 14 days before the cohabitation period, through the cohabitation period (maximum of 13 days) and continuing through the day of euthanasia (through day 25 of presumed gestation [rats that do not deliver] or day 22 of lactation [rats that deliver a litter])
F1 generation pups were not directly administered the test substance and/or the vehicle.

Frequency of treatment:

male and female: once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Dosage volume: 4 mL/kg (adjusted weekly)

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Dosage volume: 4 mL/kg (adjusted weekly)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Dosage volume: 4 mL/kg (adjusted weekly)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Dosage volume: 4 mL/kg (adjusted weekly)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on the basis of a dosage-range reproduction study
- Rationale for animal assignment: random
- Route administration rationale: The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one possible route of human exposure.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice each day

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the acclimation period and on the first day of dosage administration, weekly during the dosage period and once on the day of euthanasia (males and females), on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11, 15 and 22 (terminal body weight) (females)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption: weekly during the acclimation and dosage periods, except during cohabitation. Feed consumption values were also recorded for female rats on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11 and 15.Because pups begin to consume maternal feed on or about DL 15, feed consumption values were not tabulated after DL 15.

OTHER:
Female rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter size (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on DLs 1, 5, 8, 15 and 22.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals, after completion of the cohabitation period
- Maternal animals: All surviving animals, after completion of the 22-day postpartum period

GROSS NECROPSY
Initial physical examination of external surfaces and all orifices, as well as an internal examination of tissues and organs in situ. The following were examined: external and internal portions of all hollow organs; the external surfaces of the brain and spinal column; the nasal cavity and neck with associated organs and tissues; the thoracic, abdominal and pelvic cavities with associated organs and tissues; and the musculo/skeletal carcass. The lungs were perfused with 10% NBF.

Other examinations:

Estrous cyclicity: Evaluated daily by examination of vaginal cytology
Sperm parameters:. Parameters examined in P male parental generations:
Right testis, left testis, left epididymis (whole and cauda), right epididymis, seminal vesicles (with and without fluid) and prostate. Sperm concentration and motility were evaluated.

Statistics:

All data were tabulated, summarized and/or statistically analyzed:
-body weights, feed consumption values and percent mortality per litter: Parametric test
- litter size or the day a developmental landmark appeared: Nonparametric test
- Clinical observations and other proportion data: Variance Test for Homogeneity of the Binomial Distribution

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs in P generation male rats that were attributed to treatment with the test substance included slight, moderate and extreme excess salivation at 500 mg/kg/day. Slight excess salivation was observed as early as DS 3 and persisted until scheduled euthanasia. Slight excess salivation was observed as early as DS 3 and persisted until scheduled euthanasia.
Each of these clinical observations occurred in significantly more (p≤0.01) P generation male rats in comparison to the vehicle control group values. All P generation male rats in the 500 mg/kg/day dosage group had slight excess salivation on one or more occasions, and 20 of 25 P generation male rats in this same dosage group also had moderate excess salivation. In addition, a low incidence of ungroomed coat (N=4; p≤0.01) occurred at 500 mg/kg/day.
All other clinical observations during the precohabitation, gestation and lactation periods were considered unrelated to the test item because: 1) the incidences were not dosage-dependent; and/or 2) the number of rats affected did not differ significantly from the vehicle control group values.
The statistically significant increase (p≤0.01) in the incidence of soft and liquid feces that occurred in the 200 mg/kg/day dosage group during lactation was considered unrelated to
the test item because the increase was independent of dose.
All other clinical observations were considered unrelated to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related deaths at any dosage level tested.
One male rat in the 200 mg/kg/day dosage group was found dead approximately one hour after dosage administration on DS 116. The cause of death for the rat that died was not determined based on the in-life, postmortem and microscopic observations.
All other P generation male rats survived to scheduled euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant increase (p≤0.05) in body weight gains that occurred in P generation male rats in the 500 mg/kg/day dosage group on DSs 57 to 64 was considered unrelated to treatment with the test item because the increase was transient and did not affect the overall body weight gain.
Body weights and body weight gains during the precohabitation, gestation and lactation periods were unaffected by dosages of the test substance as high as 500 mg/kg/day. All
values were comparable among the four dosage groups.
Statistically significant increases (p≤0.01) in body weight gain occurred at 75 and 500 mg/kg/day on days 1 to 5 of lactation (days of lactation 1 to 5) and at 500 mg/kg/day on days of lactation 1 to 22, in comparison to the vehicle control group values.
The average maternal body weight on day of lactation 22 was significantly increased (p≤0.01) in the 500 mg/kg/day dosage group, as compared to the vehicle control group value. These increases in body weight and body weight gain were considered unrelated to treatment because: 1) the increase was independent of dose; 2) the increase was transient; and/or 3) the increase reflected a net loss in body weight that occurred in the vehicle control group on days of lactation 1 to 5. At 75 mg/kg/day, body weight gains were significantly reduced (p≤0.05) on days of lactation 8 to 11, in comparison to the vehicle control group value. This reduction was considered unrelated to the test item because the reduction was transient and independent of dose.
Terminal body weights for P generation male rats treated with 500 mg/kg/day of the test item were slightly reduced (by 6%), in comparison to the vehicle control group value.
This reduction did not reach statistical significance, but reflected an overall reduction (significant at p≤0.01) in body weight gains that occurred at 500 mg/kg/day for the cumulative dosage period (DSs 1 to 127).
Terminal body weights for P generation female rats treated with 500 mg/kg/day of the test item were significantly increased (p≤0.01), in comparison to the vehicle control group value.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Absolute and relative feed consumption values were unaffected by dosages of the test substance as high as 500 mg/kg/day. All values were comparable among the four dosage groups. Transient, but statistically significant increases (p≤0.05 or p≤0.01) in relative feed consumption occurred at 500 mg/kg/day on DSs 57 to 64, DSs 113 to 120 and DSs 120 to 127, in comparison to the vehicle control group values. These increases were considered unrelated to treatment with the test item because: 1) the increases were transient; and 2) there was no corresponding change in absolute feed consumption in this dosage group.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative (% terminal body weight) weights of the brain were significantly increased (p≤0.01; 4% and 11% of controls, respectively) in the 500 mg/kg/day dosage group, in comparison to the vehicle control group values.
The absolute and relative (% terminal body weight) weights of the liver (35% and 44% of controls, respectively) and spleen (17% and 25% of controls, respectively) were significantly increased (p≤0.01) at 500 mg/kg/day, in comparison to the vehicle control group values.
The absolute and relative weights of the left and right kidney were increased or significantly increased (p≤0.05 or p≤0.01; 5% to 14% of controls) at 500 mg/kg/day, as compared to the vehicle control group.
The weights of the epididymides, cauda epididymis, testes, seminal vesicles (with and without fluid) and prostate and the ratios of these organ weights to terminal body weight were unaffected by dosages of the test item as high as 500 mg/kg/day.
The statistically significant reduction (p≤0.05) in the absolute weight of the left cauda epididymis in the 500 mg/kg/day was considered unrelated to citronellyl nitrile because:
1) the reduction reflected several male rats that had a left cauda epididymis weight that was below the historical mean (0.3088 g), but was still within the range (0.1300 g to
0.3736 g) observed historically at the Testing Facility; and 2) the reduction in the weight of the left cauda epididymis occurred in the absence of reductions in the left or right
epididymis or testis.
All other statistically significant changes were considered unrelated to the test substance because:
1) the changes were independent of dose; and/or
2) thechanges were minimal and reflected normal biologic variation.
Similar to P generation male rats, the absolute and relative (% terminal body weight) weights of the liver were significantly increased (p≤0.01; 15% and 9% of controls, respectively) in the 500 mg/kg/day dosage group, in comparison to the vehicle control group values.
The absolute weights of the left and right kidney were significantly increased (p≤0.01) at 500 mg/kg/day, as compared to the vehicle control group.
There were no corresponding change in the ratio of the kidney weights to terminal body weight; however, similar observations occurred in P generation male rats in this same dosage group. In addition, there were no microscopic changes in the P generation female rats that could be correlated with the differences in liver or kidney weights.
The absolute and relative (% terminal body weight) weights of the left and right ovary and the non-gravid uterus with cervix were unaffected by dosages of the test item as high as 500 mg/kg/day.
All other statistically significant changes were considered unrelated to treatment because the changes were independent of dose.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related necropsy observations. All necropsy observations were considered unrelated to the test substance because: 1) the incidences were not
dosage-dependent; 2) the observations occurred in only one or two rats in any dosage group; or 3) they were observations that occurred in rats that died or were euthanized prior to scheduled euthanasia.
Other necropsy observations included a mass on the left lateral lobe of the liver, moderate dilation of the pelvis in the right kidney and a flaccid right testis. No other gross lesions occurred.
Rat 778 in the 500 mg/kg/day dosage group had a grossly visible reduction in the size of the epididymides, testes and left cauda epididymis. This rat also had a mottled (red and dark red) appearance to the lungs. Microscopic examination of the affected reproductive organs revealed moderate atrophy of the epididymides and left cauda epididymis and moderate atrophy of the seminiferous tubules. This rat also had a mild degree of congestion I the lungs.
There were no test substance-related necropsy observations.
All gross lesions were considered unrelated to treatment because: 1) the incidences were not dosage-dependent; or 2) the observations occurred in only one rat in any dosage group.
These necropsy observations included a mottled (red and dark red) appearance of the lungs, a dark mass on the left lateral lobe of the liver, an accessory spleen, marked pyometra and a blocked vaginal lumen. The female rat (no. 16221, vehicle control group) with the blocked vaginal lumen mated with its cohort male rat, but was not pregnant. No other gross lesions occurred in the P generation female rats.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no gross or microscopic test substance-related pathology findings observed in the rats evaluated. The changes observed in these rats were considered to be incidental or
spontaneous changes commonly observed in control Crl:CD (SD) rats. There were no adverse pathology findings in these rats related to the daily oral gavage of the test substance under the conditions of this study.

Key result

Dose descriptor:

NOAEL

Effect level:

200 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

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Organ:

other: all observed

Conclusions:

Based on the results of this study, the no-observable-adverse-effect-level (NOAEL) for toxicity of the test substance is 200 mg/kg/day. Increased incidences of excess salivation and/or ungroomed coat occured in P generation male and/or female rats in the 500 mg/kg/day dosage group. However, these clinical signs were not considered an adverse effect of the test item. At 500 mg/kg/day, sporadic reductions in body weight gains occured in male rats prior to cohabitation, followed by persistent reductions in weight gain through the end of the study. Non-reproductive organ weights were affected in both sexes at the end of the dosage period, as well as terminal body weights. Increased brain and spleen weights occured in male rats treated with 500 mg/kg/day test item, while increased liver and kidney weights occured in both sexexs in this same dosage group. The toxicological significance of the increased organ weights was unable to be determined because there were no microscopic findings that could be correlated with the changes in organ weights.

Reference 3

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Oral (Gavage) One-Generation Reproduction Study

Type of information:

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

Adequacy of study:

key study

Study period:

27 FEB 2009 - 01 FEB 2011

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:

other: OECD guidelines for the testing of chemicals. One-generation reproduction toxicity, No. 415 section 4 Health Effects (Pink pages); adopted May 26, 1983. Organisation for Economic Co-operation and Development.

Version / remarks:

Deviations see Deviation (other information):
-Acclimation
-Formulation Preparation
-Dosage Administration
-Clinical Observations
-Litter Observations
-Sexual Maturation
-Gross Necropsy, Organ Weights and Tissue Retention

Deviations:

yes

Remarks:

The deviations from the protocol had no impact on the study outcome or interpretation of the data because, relative to the total number of animals evaluated and/or the number of data points collected per parameter, the deviations were not significant.

Principles of method if other than guideline:

The requirements of the Organisation for Economic Co-operation and Development Guidelines for Testing of Chemicals No. 415 were used as the basis for study design.

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: 37 days (age at arrival), female 65 days (age at arrival)
- Weight at study initiation: (P) Males: 129 - 160 g; Females: 224 - 266 g
- Housing: individually housed in stainless steel, wire-bottomed cages; during cohabitation, each pair of male and female rats was housed in the male rat's cage; P generation female rats were individually housed in nesting boxes beginning no later than day of (presumed) gestation 20
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 19°C to 25°C
- Humidity: 30% to 70%
- Air changes: a minimum of ten changes per hour
- Photoperiod: 12-hours light:12-hours dark fluorescent light cycle
- Enrichment: supplied to all rats during the course of the study

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Results indicate that the formulations are within the acceptable limits of ±15% of nominal concentrations. The formulations are also within the acceptable limits of ≤5% RSD for homogeneity

DIET PREPARATION
Dosages were adjusted weekly for body weight changes and given at approximately the same time each day. Any dams in the process of parturition were not given the test substance and/or vehicle until the following work day. No dam missed more than one daily dosage administration, and such events were noted in the raw data. Prepared formulations were stirred continuously during dosage administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 10, 30, 100, or 300 mg/kg/day of test item
- Amount of vehicle: 4 mL/kg for 0 mg/kg/day of test item

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose formulations were analyzed by HPLC-UV.
The method was validated for the analysis of dose formulations at concentrations ranging from 1.2 mg/mL to 150 mg/mL of the test item in corn oil.
Results for all dose formulations met the acceptance criteria for concentration (within ±15% of nominal concentration) and homogeneity (≤5% relative standard deviation) except for end of study samples for Group II (18.75 mg/mL), Group III (50 mg/mL), Group IV (125 mg/mL) are out of specification (+27.3%, +20.3% and +20.0%, respectively). Time zero stability samples at 125 mg/mL were not within the acceptable limits of ±15% of nominal concentration, (+25.6%); however, the result was accepted and used as the reference concentration for formulation stability evaluations.

Duration of treatment / exposure:

male: once daily beginning 83 days before the cohabitation period, through the cohabitation period (maximum of 13 days) and continuing until the day before euthanasia
female: once daily beginning 14 days before the cohabitation period, through the cohabitation period (maximum of 13 days) and continuing through the day of euthanasia (through day 25 of presumed gestation [rats that do not deliver] or day 22 of lactation [rats that deliver a litter])
F1 generation pups were not directly administered the test substance and/or the vehicle.

Frequency of treatment:

male and female: once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Dosage volume: 4 mL/kg (adjusted weekly)

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Dosage volume: 4 mL/kg (adjusted weekly)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Dosage volume: 4 mL/kg (adjusted weekly)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Dosage volume: 4 mL/kg (adjusted weekly)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on the basis of a dosage-range reproduction study
- Rationale for animal assignment: random
- Route administration rationale: The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one possible route of human exposure.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice each day

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the acclimation period and on the first day of dosage administration, weekly during the dosage period and once on the day of euthanasia (males and females), on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11, 15 and 22 (terminal body weight) (females)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption: weekly during the acclimation and dosage periods, except during cohabitation. Feed consumption values were also recorded for female rats on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11 and 15.Because pups begin to consume maternal feed on or about DL 15, feed consumption values were not tabulated after DL 15.

OTHER:
Female rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter size (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on DLs 1, 5, 8, 15 and 22.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals, after completion of the cohabitation period
- Maternal animals: All surviving animals, after completion of the 22-day postpartum period

GROSS NECROPSY
Initial physical examination of external surfaces and all orifices, as well as an internal examination of tissues and organs in situ. The following were examined: external and internal portions of all hollow organs; the external surfaces of the brain and spinal column; the nasal cavity and neck with associated organs and tissues; the thoracic, abdominal and pelvic cavities with associated organs and tissues; and the musculo/skeletal carcass. The lungs were perfused with 10% NBF.

Other examinations:

Estrous cyclicity: Evaluated daily by examination of vaginal cytology
Sperm parameters:. Parameters examined in P male parental generations:
Right testis, left testis, left epididymis (whole and cauda), right epididymis, seminal vesicles (with and without fluid) and prostate. Sperm concentration and motility were evaluated.

Statistics:

All data were tabulated, summarized and/or statistically analyzed:
-body weights, feed consumption values and percent mortality per litter: Parametric test
- litter size or the day a developmental landmark appeared: Nonparametric test
- Clinical observations and other proportion data: Variance Test for Homogeneity of the Binomial Distribution

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs in P generation male rats that were attributed to treatment with the test substance included slight, moderate and extreme excess salivation at 500 mg/kg/day. Slight excess salivation was observed as early as DS 3 and persisted until scheduled euthanasia. Slight excess salivation was observed as early as DS 3 and persisted until scheduled euthanasia.
Each of these clinical observations occurred in significantly more (p≤0.01) P generation male rats in comparison to the vehicle control group values. All P generation male rats in the 500 mg/kg/day dosage group had slight excess salivation on one or more occasions, and 20 of 25 P generation male rats in this same dosage group also had moderate excess salivation. In addition, a low incidence of ungroomed coat (N=4; p≤0.01) occurred at 500 mg/kg/day.
All other clinical observations during the precohabitation, gestation and lactation periods were considered unrelated to the test item because: 1) the incidences were not dosage-dependent; and/or 2) the number of rats affected did not differ significantly from the vehicle control group values.
The statistically significant increase (p≤0.01) in the incidence of soft and liquid feces that occurred in the 200 mg/kg/day dosage group during lactation was considered unrelated to
the test item because the increase was independent of dose.
All other clinical observations were considered unrelated to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related deaths at any dosage level tested.
One male rat in the 200 mg/kg/day dosage group was found dead approximately one hour after dosage administration on DS 116. The cause of death for the rat that died was not determined based on the in-life, postmortem and microscopic observations.
All other P generation male rats survived to scheduled euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant increase (p≤0.05) in body weight gains that occurred in P generation male rats in the 500 mg/kg/day dosage group on DSs 57 to 64 was considered unrelated to treatment with the test item because the increase was transient and did not affect the overall body weight gain.
Body weights and body weight gains during the precohabitation, gestation and lactation periods were unaffected by dosages of the test substance as high as 500 mg/kg/day. All
values were comparable among the four dosage groups.
Statistically significant increases (p≤0.01) in body weight gain occurred at 75 and 500 mg/kg/day on days 1 to 5 of lactation (days of lactation 1 to 5) and at 500 mg/kg/day on days of lactation 1 to 22, in comparison to the vehicle control group values.
The average maternal body weight on day of lactation 22 was significantly increased (p≤0.01) in the 500 mg/kg/day dosage group, as compared to the vehicle control group value. These increases in body weight and body weight gain were considered unrelated to treatment because: 1) the increase was independent of dose; 2) the increase was transient; and/or 3) the increase reflected a net loss in body weight that occurred in the vehicle control group on days of lactation 1 to 5. At 75 mg/kg/day, body weight gains were significantly reduced (p≤0.05) on days of lactation 8 to 11, in comparison to the vehicle control group value. This reduction was considered unrelated to the test item because the reduction was transient and independent of dose.
Terminal body weights for P generation male rats treated with 500 mg/kg/day of the test item were slightly reduced (by 6%), in comparison to the vehicle control group value.
This reduction did not reach statistical significance, but reflected an overall reduction (significant at p≤0.01) in body weight gains that occurred at 500 mg/kg/day for the cumulative dosage period (DSs 1 to 127).
Terminal body weights for P generation female rats treated with 500 mg/kg/day of the test item were significantly increased (p≤0.01), in comparison to the vehicle control group value.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Absolute and relative feed consumption values were unaffected by dosages of the test substance as high as 500 mg/kg/day. All values were comparable among the four dosage groups. Transient, but statistically significant increases (p≤0.05 or p≤0.01) in relative feed consumption occurred at 500 mg/kg/day on DSs 57 to 64, DSs 113 to 120 and DSs 120 to 127, in comparison to the vehicle control group values. These increases were considered unrelated to treatment with the test item because: 1) the increases were transient; and 2) there was no corresponding change in absolute feed consumption in this dosage group.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative (% terminal body weight) weights of the brain were significantly increased (p≤0.01; 4% and 11% of controls, respectively) in the 500 mg/kg/day dosage group, in comparison to the vehicle control group values.
The absolute and relative (% terminal body weight) weights of the liver (35% and 44% of controls, respectively) and spleen (17% and 25% of controls, respectively) were significantly increased (p≤0.01) at 500 mg/kg/day, in comparison to the vehicle control group values.
The absolute and relative weights of the left and right kidney were increased or significantly increased (p≤0.05 or p≤0.01; 5% to 14% of controls) at 500 mg/kg/day, as compared to the vehicle control group.
The weights of the epididymides, cauda epididymis, testes, seminal vesicles (with and without fluid) and prostate and the ratios of these organ weights to terminal body weight were unaffected by dosages of the test item as high as 500 mg/kg/day.
The statistically significant reduction (p≤0.05) in the absolute weight of the left cauda epididymis in the 500 mg/kg/day was considered unrelated to citronellyl nitrile because:
1) the reduction reflected several male rats that had a left cauda epididymis weight that was below the historical mean (0.3088 g), but was still within the range (0.1300 g to
0.3736 g) observed historically at the Testing Facility; and 2) the reduction in the weight of the left cauda epididymis occurred in the absence of reductions in the left or right
epididymis or testis.
All other statistically significant changes were considered unrelated to the test substance because:
1) the changes were independent of dose; and/or
2) thechanges were minimal and reflected normal biologic variation.
Similar to P generation male rats, the absolute and relative (% terminal body weight) weights of the liver were significantly increased (p≤0.01; 15% and 9% of controls, respectively) in the 500 mg/kg/day dosage group, in comparison to the vehicle control group values.
The absolute weights of the left and right kidney were significantly increased (p≤0.01) at 500 mg/kg/day, as compared to the vehicle control group.
There were no corresponding change in the ratio of the kidney weights to terminal body weight; however, similar observations occurred in P generation male rats in this same dosage group. In addition, there were no microscopic changes in the P generation female rats that could be correlated with the differences in liver or kidney weights.
The absolute and relative (% terminal body weight) weights of the left and right ovary and the non-gravid uterus with cervix were unaffected by dosages of the test item as high as 500 mg/kg/day.
All other statistically significant changes were considered unrelated to treatment because the changes were independent of dose.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related necropsy observations. All necropsy observations were considered unrelated to the test substance because: 1) the incidences were not
dosage-dependent; 2) the observations occurred in only one or two rats in any dosage group; or 3) they were observations that occurred in rats that died or were euthanized prior to scheduled euthanasia.
Other necropsy observations included a mass on the left lateral lobe of the liver, moderate dilation of the pelvis in the right kidney and a flaccid right testis. No other gross lesions occurred.
Rat 778 in the 500 mg/kg/day dosage group had a grossly visible reduction in the size of the epididymides, testes and left cauda epididymis. This rat also had a mottled (red and dark red) appearance to the lungs. Microscopic examination of the affected reproductive organs revealed moderate atrophy of the epididymides and left cauda epididymis and moderate atrophy of the seminiferous tubules. This rat also had a mild degree of congestion I the lungs.
There were no test substance-related necropsy observations.
All gross lesions were considered unrelated to treatment because: 1) the incidences were not dosage-dependent; or 2) the observations occurred in only one rat in any dosage group.
These necropsy observations included a mottled (red and dark red) appearance of the lungs, a dark mass on the left lateral lobe of the liver, an accessory spleen, marked pyometra and a blocked vaginal lumen. The female rat (no. 16221, vehicle control group) with the blocked vaginal lumen mated with its cohort male rat, but was not pregnant. No other gross lesions occurred in the P generation female rats.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no gross or microscopic test substance-related pathology findings observed in the rats evaluated. The changes observed in these rats were considered to be incidental or
spontaneous changes commonly observed in control Crl:CD (SD) rats. There were no adverse pathology findings in these rats related to the daily oral gavage of the test substance under the conditions of this study.

Key result

Dose descriptor:

NOAEL

Effect level:

200 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

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Organ:

other: all observed

Conclusions:

Based on the results of this study, the no-observable-adverse-effect-level (NOAEL) for toxicity of the test substance is 200 mg/kg/day. Increased incidences of excess salivation and/or ungroomed coat occured in P generation male and/or female rats in the 500 mg/kg/day dosage group. However, these clinical signs were not considered an adverse effect of the test item. At 500 mg/kg/day, sporadic reductions in body weight gains occured in male rats prior to cohabitation, followed by persistent reductions in weight gain through the end of the study. Non-reproductive organ weights were affected in both sexes at the end of the dosage period, as well as terminal body weights. Increased brain and spleen weights occured in male rats treated with 500 mg/kg/day test item, while increased liver and kidney weights occured in both sexexs in this same dosage group. The toxicological significance of the increased organ weights was unable to be determined because there were no microscopic findings that could be correlated with the changes in organ weights.

Reference 4

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Specific target organ toxicity - repeated exposure

Target organ toxicity (repeated exposure) means specific, target organ toxicity arising from a repeated exposure to a substance or mixture. All significant health effects that can impair function, both reversible and irreversible, immediate and/or delayed are included.

An oral (gavage) 90-day repeated dose study in the rat (OECD 408) and a one-generation reproduction toxicity study have been used to assess the repeated dose toxicity of the substance.

In the OECD 408 study, oral administration of the substance to rats by gavage, at dose levels 300, 100, 30 and 10 mg/kg bw/day, resulted in no treatment related adverse effects at any concentration. The 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day.

In the one-generation reproduction toxicity study, based on OECD test guideline 415, oral administration of the substance to rats by gavage, at dose levels 500, 200 and 75 mg/kg bw/day resulted in At 500 mg/kg/day, sporadic reductions in body weight gains occurred in male rats prior to cohabitation, followed by persistent reductions in weight gain through the end of the study. Non-reproductive organ weights were affected in both sexes at the end of the dosage period, as well as terminal body weights. Increased brain and spleen weights occurred in male rats treated with 500 mg/kg/day test item, while increased liver and kidney weights occurred in both sexes in this same dosage group. The toxicological significance of the increased organ weights was unable to be determined because there were no microscopic findings that could be correlated with the changes in organ weights. The 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was considered to be 200 mg/kg/day.

No significant/adverse toxic effects were therefore observed within the classification guidance value ranges in the CLP regulation for specific target organ toxicity-repeated exposure (Category 2) based on a 90-day exposure.i. e. no significant/adverse toxic effects were seen at or below a dose of 100 mg/kg bw/day. The effects observed in the 90 -day and one generation reproductive toxicity study were not considered to be signs of significant toxicity and changes in organ weights were not accompanied by evidence of organ dysfunction.

Based on these results, the substance is not classified for specific target organ toxicity-repeated exposure.