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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-03-20 to 2007-10-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study READ ACROSS To address toxicological endpoints as part of the REACH registration of Lemonile (Target Substance) it is proposed to read-across to Citronellyl Nitrile (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised in using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore read across is justified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Remarks:
date of inspection 2005-08-30; date of signature 2005-11-05
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Citronellyl nitrile
- Molecular formula: C10H17N
- Molecular weight: 151.25 Da
- Smiles notation: N#CCC(CC/C=C(/C)C)C
- InChl: InChI=1S/C10H17N/c1-9(2)5-4-6-10(3)7-8-11/h5,10H,4,6-7H2,1-3H3
- Structural formula attached as image file (see illustration below).
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Analytical purity: 99.8 % (sum of isomers)
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components:
- Isomers composition: 96.8 % cis isomer (therefore 3.0 % trans isomer)
- Purity test date: 2007-02-28

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, United Kingdom
- Age at study initiation: approx. 5 wk to 8 wk
- Weight at study initiation: Males 147 g to 183 g, females 131 g to 162 g
- Fasting period before study: No data
- Housing: In groups of 3 or 4 by sex in polypropylene grid-floor cages
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature 21 °C ± 2 °C
- Humidity: 55 % ± 15 %; occasional deviations that did not affect study.
- Air changes: 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentrations as a solution in corn oil. Formulations were prepared weekly and stored at 4 °C in the dark.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of citronellyl nitrile in the test material formulations was determined by gas chromatography (GC) using an external standard technique.

GC system: Agilent Technologies 5890, incorporating autosampler and workstation
Column: DB-5 (30 m x 0.25 mm id x 0.25 µm film)
Oven temperature program:
- initial: 100 °C for 0 mins
- rate: 10 °C/min
- temp: 150 °C for 0 mins
- rate: 70 °C/min
- final: 325 °C for 3 mins
Injection temperature: 250 °C
Flame ionisation detector temperature: 300 °C
Injection volume: 1 µL
Retention time: ~4.2 mins
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on previous toxicity data
- Rationale for animal assignment: Random
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGESIDE OBSERVATIONS
- Time schedule: Immediately before and after dosing, and 1 hr and 5 hr after dosing during the work week. Immediately before dosing and 1 hr after dosing at weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Prior to start of treatment, weekly thereafter.
- Observations performed: Gait, tremors, twitches, convulsions, bizarre/abnormal stereotypic behaviour, salivation, piloerection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.

BODY WEIGHT:
- Time schedule for examinations: Day 1, weekly thereafter and at terminal kill.

FOOD CONSUMPTION:
- Food consumption for each group determined and mean daily diet consumption calculated as g food/kg body weight/day.

FOOD EFFICIENCY:
- For each group, percentage body weight gain in kg/food consumption in kg per week calculated from consumption and body weight gain data.

WATER CONSUMPTION
- Time schedule for examinations: Daily for each cage group by visual inspection of the water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: Pre-treatment and before termination of treatment (wk 12)
- All control and dose animals examined.

HAEMATOLOGY
- Time schedule for collection of blood: During wk 7 and at end of study (90 d)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All animals
- Parameters examined: Haemoglobin, erythrocyte count, haemocrit, erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, reticulocyte count (cresyl blue stained slides were prepared but reticulocytes were not assessed).

CLINICAL CHEMISTRY
- Time schedule for collection of blood: During wk 7 and at end of study (90 d)
- Animals fasted: No
- How many animals: All animals
- Parameters examined: Urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin.

URINALYSIS
- Time schedule for collection of urine: During wk 7 and at end of study (90 d)
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: Volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances, blood

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: Daily, but only half animals tested each day (alternating by sex).
- Dose groups that were examined: All
- Battery of functions tested: sensory activity, grip strength, motor activity

OESTROUS CYCLES
- Time schedule for examination: During wk 6 and wk 7, and wk 12 and wk 13
- Examination performed: Vaginal smear taken daily and sample was placed on glass slide. Smears allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of oestrous recorded.
Sacrifice and pathology:
GROSS PATHOLOGY
- All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were detected.

HISTOPATHOLOGY
- Samples of the following tissues were removed from each animal and preserved in buffered 10 % formalin: adrenals, aorta (thoracic), bone and bone marrow (femur including stifle joint), bone and bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, right epididymis, eyes, gross lesions, heart, ileum (including Peyer's patches), jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary glands, muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal chord (cervical, mid-thoracic and lumbar), spleen, stomach, right testis, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus.
- Control and 300 mg/kg bw/d dose group animals prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examinations. Any macroscopically observed lesion was also processed.
- Due to indications of treatment-related bone marrow and liver changes, examination was subsequently extended to include similarly-prepared sections from all animals in the other treatment groups.

SEMEN EXAMINATIONS
- At necropsy the following evaluations were performed on all males:
-- 1. The left testis and epididymis were removed, dissected from connective tissue and weighed separately.
-- 2. For testis, tunica albuginea were removed and the testicular tissue stored frozen at approx. -20 °C. At a later date, the samples were thawed, reweighed and homogenised in a suitable saline/detergent mixture. Samples of the homogenate were stained with a DNA-specific fluorescent stain and a subsample was analysed for numbers of homogenisation resistant spermatids.
-- 3. For the epididymis, the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility and sperm morphology. A minimum of 200 individual sperm were assessed using an automated semen analyser to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).
-- 4. A sample of semen was preserved in formalin and then stained with eosin. A subsample was placed on a glass slide with a coverslip and a morphometric analysis of sampled semen was performed.
-- 5. The cauda epididymis was separated from the body of the epididymis and weighed. The cauda epididymis was then frozen at approx. -20 °C. At a later date, the samples were thawed, reweighed and homogenised in an appropriate saline/detergent mixture. Samples of the homogenate were stained with eosin and a subsample was analysed for homogenisation resistant spermatids.

For both 2 and 4 above, samples from groups 1 and 5 (i.e. control and 300 mg/kg bw/d respectively) were examined only as no significant effects were seen.
Statistics:
Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of the means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data, or the Shirley Test for non-parametric data. If no dose-response was found, but the data showed non-homogeneity of means, the data were analysed using a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine the differences from the control group. Finally, if required, pairwise tests were performed using Student's t-test (parametric) or Mann-Whitney test (non-parametric).

Histopathological data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes.
- χ² for differences in the frequency of lesions occurring with an overall frequency of 1 or greater.
- Kruskal-Wallis 1-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
wk 7, wk 13, increase in protein content in females fed 300 mg/kg bw/d. Wk 13 Increase in albumin content in males fed 300 mg/kg bw/d.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals of both sexes treated with 300 mg/kg bw/d showed statistically-significant increase in liver weight both absolute and relative to terminal body weight. Males fed 100 mg/kg bw/d also showed effect on liver weight.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
Details on results:
NB: Tables and appendices are included in the supporting information section below.

MORTALITY
- See table 1
- There were no unscheduled deaths

CLINICAL SIGNS
- See tables 2 and 3.
- 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 mg/kg bw/d animals throughout the treatment period. Incidents of increased salivation were also evident in 100, 30, 10 mg/kg bw/d 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 systematic toxicity.
- Isolated instances of generalised fur loss, wet fur, wounds, scab formation or red/brown stained snout were evidence 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 41 d and 60 d and an abnormal gait between 41 d and 49 d. These were isolated incidental findings unrelated to treatment.

BEHAVIOURAL ASSESSMENT
- There were no treatment related changes in the behavioural parameters measured.
- All inter- and intra-group differences in behavioural scores were considered to be part of normal variation for rats of the strain and age used and were of no toxicological importance.

FUNCTIONAL PERFORMANCE TESTS
- There were no toxicologically significant changes in the functional performance parameters measured.
- Males treated with 300 and 100 mg/kg bw/d showed a statistically significant reduction in overall activity whilst females treated with 10 mg/kg bw/d showed a statistically significant increase in overall activity. Males treated with 100 mg/kg bw/d also showed a statistically significant reduction in the last 20 % of activity. In the absence of a dose-related response or any supporting clinical observations to suggest an effect of neurotoxicity, these findings were considered to be of no toxicological significance.
- Females treated with 300 and 100 mg/kg bw/d showed a statistically significant increase in mean hind limb grip strength. This effect was confined to 1 out of the 3 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.

SENSORY REACTIVITY ASSESSMENTS
- There were no treatment-related changes in sensory activity.
- All inter- and intra-group differences were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

BODY WEIGHT AND WEIGHT GAIN
- See table 6, figures 1 and 2.
- Bodyweight gain in test animals during treatment period was similar to that of controls.
- Females treated with 10 mg/kg bw/d showed a statistically-significant increase in bodyweight gain in wk 3 whilst females treated with 300, 100 and 30 mg/kg bw/d showed statistically significant reduction in bodyweight gain in wk 8. In the absence of dose-related response the intergroup differences were considered to be of no toxicological importance.

FOOD CONSUMPTION AND COMPOUND INTAKE
- See table 8, figure 4.
- No adverse effect on food consumption during the study period.

FOOD EFFICIENCY
- See table 9.
- Food efficiency (ratio of bodyweight gain to dietary intake) similar to that of controls.

WATER CONSUMPTION
- Daily visual inspection of water bottles revealed no intergroup differences.

OPHTHALMOSCOPIC EXAMINATION
- See appendix 6.
- There were no treatment-related effects. Incidental findings were those normally encountered in laboratory-maintained rats of this age and strain.

HAEMATOLOGY
- See table 10, appendices 7 and 8.
- There were no toxicologically significant changes in haematological parameters measured.
- During wk 7 assessment, males from all treated groups showed a statistically significant reduction in haemoglobin. Males treated with 300 and 100 mg/kg bw/d also showed a reduction in mean cell volume and neutrophils. In the absence of dose-related response these intergroup differences were considered of no toxicological importance.
- Males treated with 300 and 100 mg/kg bw/d showed a statistically significant reduction in mean cell haemoglobin and an increase in lymphocyte count (300 mg/kg bw/d only). Reductions in mean cell haemoglobin were still evident during wk 13 assessments together with statistically significant reductions in mean cell haemoglobin concentration for 300 mg/kg bw/d males, haemocrit levels for 100 mg/kg bw/d males and an increase in erythrocyte count for 300 mg/kg bw/d. 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 CHEMISTRY
- See table 11, appendix 9.
- During wk 7 assessment, a statistically significant increase in total protein was evident for females treated with 300 mg/kg bw/d
- Increases in total protein were still evident during wk 13 assessments for females treated with 300 mg/kg bw/d. Males from this treatment level also showed a statistically significant increase in albumin levels.
- No such effects were detected in animals of either sex at 100, 30 or 10 mg/kg bw/d
- During wk 7, males from all treated groups showed a statistically significant reduction in alanine aminotransferase. In the absence of 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 mg/kg bw/d showed a statistically significant reduction in albumin/globulin ratio during wk 7 assessments. This intergroup difference was considered to be higher than normal control values and was considered of no toxicological significance.

URINALYSIS
- See table 12, appendix 10
- No treatment-related effects were detected in the urinanalytical parameters measured.

OESTROUS CYCLE ASSESSMENTS
- See table 13, appendix 11.
- There were no treatment-related effects on female oestrous cycles or on type or proportion of females with abnormal oestrous cycles.

ORGAN WEIGHTS
- See table 14, appendices 12 and 13.
- Animals of both sexes treated with 300 mg/kg bw/d showed a statistically significant increase in liver weight both absolute and relative to terminal bodyweight. The effect of liver weight also extended to males treated with 100 mg/kg bw/d.
- No such effects were detected in females on 100 mg/kg bw/d or in animals of either sex treated with 30 or 10 mg/kg bw/d.
- Males treated with 100 mg/kg bw/d showed a statistically significant increase in heart 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 of no toxicological significance.

GROSS PATHOLOGY
- See table 15, appendix 14.
- No toxicologically significant macroscopic abnormalities were detected.
- Hydronephrosis was evidence in 1 male treated with 300 mg/kg bw/d and in 2 males and 1 female treated with 30 mg/kg bw/d. 1 male treated with 30 mg/kg bw/d also showed small and flaccid testes whilst 2 females treated with 300 mg/kg bw/d showed in the ovaries, a fluid fill sac. Finally, lung changes (dark, pale or red lobes) were evidence in 1 male and 1 female treated with 10 or 100 mg/kg bw/d. In the absence of a dose-related response or any histological correlates the intergroup differences were considered of no toxicological importance.

SPERM ANALYSIS
- See tables 16, 17, and 18, appendices 15, 16 and 17.
- There were no treatment related effects detected in sperm motility values, morphological assessments or in homogenisation-resistant spermatid counts.

HISTOPATHOLOGY: NON-NEOPLASTIC
- See table 19, appendix 18.
- Liver:
-- Marginal centrilobar hepatocyte enlargement was observed among animals of either sex treated with 300 mg/kg bw/d (p < 0.05). 2 instances of centrilobular hepatocyte enlargement were seen among males and one female treated with 100 mg/kg bw/d. 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 amongst 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 mg/kg bw/d, but not at any other treatment level. This was considered to be a marginal effect that did not attain statistical significance.
- All other findings 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 no toxicological significance.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

To address toxicological endpoints as part of the REACH registration of Lemonile (Target Substance) it is proposed to read-across to Citronellyl Nitrile (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised in using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore read across is justified.

See section 13, document Read across justification_Citronellyl ntrile.

Applicant's summary and conclusion

Conclusions:
The oral administration of citronellyl nitrile to rats for a period of 90 consecutive days at dose levels of 10, 30, 100 and 300 mg/kg/day 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 mg/kg/day. The NOEL for citronellyl nitrile was therefore considered to be 30 mg/kg/day.
The liver changes identified histopathologically were confined to adaptive liver changes and in isolation were considered not to represent "serious damage" to health. The bone marrow changes seen in 300 mg/kg/day females were marginal and not adverse since there was no evidence of corresponding haematological changes in females. For these reasons, 300 mg/kg/day may be regarded as a NOAEL for citronellyl nitrile.