Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-02-16 to 2011-01-25
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:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes
Remarks:
no certificate
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Names of test material (as cited in study report): Citronellyl nitrile; 3,7-Dimethyl-6-octenenitrile
- 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: Yellowish liquid/ clear colourless to yellowish liquid
- Physical state: Liquid
- Lot Nos: R7467, R8233
- Expiration dates of the lots: 2010-09, 2011-01
- Stability under storage conditions: Measured as a 157.0 mg/mL to 155.8 mg/mL (-0.8 %) over a period of 17 days
- Storage condition of test material: Room temperature, protected from light

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, United States
- Age at study initiation: Male (P) 37 d, female (P) 65 d
- Weight at study initiation: (P) Males: 129-160 g; Females: 224-266 g
- Housing:
-- P: Individually in stainless steel, wire-bottomed cages except during cohabitation and postpartum periods.
-- P: During cohabitation, each pair of male and female rats was housed in the male rat's cage.
-- P: Beginning no later than DG 20, P generation female rats were individually housed in nesting boxes until they either naturally delivered litters or were euthanised on DG 25.
-- P: Each dam and delivered litter was housed in a common nesting box during the postpartum period
-- F1 pups selected for continued evaluation after weaning were individually housed in stainless steel, wire-bottomed cages.
-- F1 pups selected for continued observation that had a body weight of < 28 g were pair housed for 1 wk in a nesting box to optimise the conditions under which pups were transitioned from group to individual housing.
- Diet: Ad libitum Certified Rodent Diet #5002 meal (PMI Nutrition International, Inc., St Louis, Missouri, United States). Analysis performed (no known contaminants).
- Water: Ad libitum, reverse osmosised. Analysis performed (no known contaminants).
- Acclimation period: Males 6 d, females 12 d

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 to 70 %.
- Air changes: 10 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Volume of vehicle: 4 mL/kg bw/day, adjusted weekly on basis of individual body weights.
- Lot nos: 058K0070; J-145
Details on mating procedure:
- M:F ratio per cage: 1:1 (except male rat 777 which cohabited with two females due to unscheduled euthanasia of male rat 796)
- Length of cohabitation: maximum of 13 d
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration and homogeneity of the prepared formulations were verified. Quadruplicate samples were taken from the top, middle and bottom of each concentration on the first day of preparation, and on the last day of preparation. Backup samples were also taken, and in the case of the last day of preparation analysed because of instrument failure on the first sample.
Duration of treatment / exposure:
- Males: Beginning 83 before the cohabitation period, throughout the cohabitation period (maximum 13 days), and continuing until the day before euthanasia.
- Females: Beginning 14 days before the cohabitation period, throughout the cohabitation period (maximum 13 days), continuing through the day of euthanasia (through day 25 of presumed gestation (rats that did not deliver) or day 22 of lactation (rats that delivered a litter)).
-- NB: 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.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
75 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were selected on the basis of a dosage-range reproduction study (TIF00043). Eight rats per sex per group were dosed via gavage with 0 (Vehicle), 10, 30, 100, or 300 mg/kg/day citronellyl nitrile in a corn oil vehicle. Dosing occurred 14 d prior to cohabitation, through cohabitation, gestation, and postpartum day 5 or the day of euthanasia. Absolute and relative feed consumption values were reduced in males from the 300 mg/kg bw/day dosage group. There were no clinical observations, body weight, body weight gain, mating or fertility parameters, or organ weight changes considered related to treatment in the male rats. In the female rats there were no clinical observations, body weight, body weight gain, feed consumption, estrous cycling, mating or fertility parameters, or natural delivery observations considered related to treatment. Based on these data, a higher dose of 500 mg/kg bw/day was selected to ensure that sufficient toxicity was observed.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
- Time schedule (both parental animals and F1 animals): Twice daily

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily before dosage administration, between 1-2 hrs following dosage administration, at end of normal working day, at euthanasia. Occasionally more frequently.
- Parameters examined: clinical signs, abortions, premature deliveries and deaths.

BODY WEIGHT:
- Time schedule for examinations: At least weekly during acclimation period, weekly during dosage period, at euthanasia.

FOOD CONSUMPTION
- Food consumption for each animal determined weekly during the accclimation and dosage periods, but not 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.
Oestrous cyclicity (parental animals):
Estrous cycling was evaluated daily by examination of vaginal cytology beginning 14 days before scheduled cohabitation and continuing through chabitation until mating was confirmed.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight (left epididymis whole and cauda), seminal vesicles (with and without fluid) and prostate. Sperm concentration and motility were also evaluated.
Litter observations:
STANDARDISATION OF LITTERS: Not performed
- Litters were not culled during the lactation period, because random selection of pups for culling could result in potential biases in pup viabilities and body weight gains during this period.

PREWEANING OBSERVATIONS
- Litters were examined after delivery to identify the number and sex of pups, stillbirths, live births and gross alterations.
- Each litter was evaluated for viability twice daily and the pups in each litter counted once daily.
- Clinical observations were recorded once daily during the preweaning period.
- Pup body weights were recorded on days 1 (birth), 5, 8, 11, 15 and 22 postpartum.
- Anogenital distance was measured for all live F1 generation pups on days 1 and 22 postpartum.
- Nipple eruption was evaluated for all live F1 generation pups once on day 12 postpartum.

POSTWEANING OBSERVATIONS
- Rats were observed for viability twice daily and for clinical observations once daily during the postweaning period.
- Body weights were recorded weekly during the postweaning period and on the day euthanasia occurred.
- Feed consumption values were recorded weekly during the postweaning period (only for individually housed rats).

Postmortem examinations (parental animals):
MALE RATS
- Sacrifice: All surviving animals, after completion of cohabitation period.
- Gross necropsy of the thoracic, abdominal and pelvic viscera was performed
- The following organs were individually weighed: 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 using computer-assisted sperm analysis (CASA). Motility was evaluated by the Hamilton Thorne IVOS by collection of a sample from the left and right vas deferens. A homogenate was prepared from the left cauda epididymis for evaluation by the Hamilton Thorne IVOS to determine sperm concentration/density (sperm per gram of tissue weight).
- The remaining portion of the left epididymis (corpus and caput), as well as the right epididymis, prostate and seminal vesicles were fixed in 10% NBF for microscopic evaluation. The testes were fixed in Bouin's solution for 48 to 96 hours and then retained in 10 % NBF for microscopic evaluation

FEMALE RATS
- Sacrifice:
-- All surviving animals, after completion of 22 day postpartum period.
-- Females that did not deliver a litter were euthanised on DG 25.

EARLY DEATHS (P-GENERATION RATS)
- Male rats that died or were euthanised before scheduled termination were examined for the cause of death or condition on the day the observation was made.
- The rats were examined for gross lesions.
- Tissues were weighed and retained, and microscopic evaluations were conducted as described below .
- The lungs, trachea and oesophagus were perfused and saved in 10 % NBF (neutral buffered formalin) for microscopic evaluation as described below.
- The heart, liver, kidneys, stomach and spleen were also retained in 10 % NBF

HISTOPATHOLOGY / ORGAN WEIGHTS
- Tissues to be examined histologically were routinely processed, embedded in paraffin, sectioned at 5 microns and stained with hematoxylin and eosin. Microscopic examination was performed on all control and high test substance dosage group P generation rats. All gross lesions were examined microscopically.
- The tissues indicated in Table "Histopathology" (see under "any other information on materials and methods, including tables" below) were prepared for microscopic examination and weighed, respectively.
- Each ovary was embedded in separate paraffin blocks. Consistency in orientation of each ovary was imperative, such that one block number was designated for ovary 1 the left ovary, and the other designated for ovary 2 the right ovary. Beginning at 200 microns within the ovary, five sections were taken 100 microns apart. All five sections were mounted on one slide beginning at the labelled end of the slide. The slide was stained with haematoxylin and eosin (H&E). Each section was quantitatively evaluated for primordial follicles, to include small growing follicles, and each animal was evaluated for presence or absence of corpora lutea of lactation. Follicle counts were reported per ovary, and as a total (both ovaries combined). After the five sections were taken from each ovary for follicular enumeration, a section of each ovary was taken and processed for histopathology. Tissues were shipped at ambient conditions to Charles River Laboratories Pathology Associates, Frederick, Maryland, Unites States for ovarian follicle analysis.
Postmortem examinations (offspring):
SACRIFICE
- F1 offspring not selected for continued evaluation were sacrificed at 22 d postpartum.
- F1 offspring selected for continued evaluation were sacrificed at 60 (± 3) d postpartum

GROSS NECROPSY
- Gross necropsy consisted of an 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
- Adult rats were examined for gross lesions. Gross lesions were retained in 10 % NBF and shipped to the Principal Investigator for histological examination.

HISTOPATHOLOGY: PUPS
- Pups that died before examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sunk were identified as stillborn; pups with lungs that floated were identified as liveborn and to have died shortly after birth. Pups with gross lesions were preserved in Bouin's solution for possible future evaluation.
- Pups that died or were euthanised before scheduled termination were examined for gross lesions and the cause of death or condition on the day the observation was made. Pups found on days 2 to 5 postpartum were preserved in Bouin's solution for possible future evaluation; pups found on days 6 to 22 postpartum were preserved in 10 % NBF.
- All pups not selected for continued evaluation were euthanised by carbon dioxide asphyxiation on day 22 postpartum and examined for gross lesions; gross lesions were preserved in 10 % NBF for possible future histopathological evaluation. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Carcasses of pups not selected for continued observation were discarded without further evaluation.


Statistics:
All data were tabulated, summarised and/or statistically analysed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft Excel, Quattro Pro 8 and The SAS System.

Variables with interval or ratio scales of measurement, such as body weights, feed consumption values and percent mortality per litter were analysed as described under the Parametric heading of the schematic. Bartlett’s Test of Homogeneity of Variances was used to estimate the probability that the dosage groups have different variances. A non-significant result (p > 0.001) indicated that an assumption of homogeneity of variance was not inappropriate, and the data were compared using the Analysis of Variance. If that test was significant (p ≤ 0.05), the groups given the test substance were compared with the control group using Dunnett’s Test. If Bartlett’s Test was significant (p ≤ 0.001), the Analysis of Variance Test was not appropriate, and the data were analysed as described under the Nonparametric heading of the schematic. When 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis Test was used to analyse the data, and in the event of a significant result (p ≤ 0.05), Dunn’s Test was used to compare the groups given the test substance with the control group. When more than 75 % of the scores in any dosage group were tied, Fisher’s Exact Test was used to compare the proportion of ties in the dosage group.

Variables that had graded our count scores, such as litter size or the day a developmental landmark appeared, were analysed using the procedures described under the Nonparametric heading of the schematic.

Clinical observations and other proportion data were analysed using the Variance Test for Homogeneity of the Binomial Distribution.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MALE MORTALITY
- There were no treatment-related deaths at any dosage level tested. One P generation male rat in each of the vehicle control, 200 and 500 mg/kg/day dosage group was humanely euthanized on DS 73, 86 or 121 as a result of a suspected gavage accident or a broken palate. A total of 72 to 121 dosages of the test substance or vehicle had been administered to these rats prior to euthanasia. In addition, 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. None of these deaths were attributed to treatment with the test substance or vehicle. Clinical and necropsy observations for these rats are provided in Text Table 3, and microscopic observations are summarized in Text Table 4.

All other P generation male rats survived to scheduled euthanasia.

MALE CLINICAL OBSERVATIONS
Clinical signs in P generation male rats that were attributed to treatment with citronellyl nitrile 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. 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 were considered unrelated to treatment with citronellyl nitrile. These clinical observations included sparse hair coat (head, neck, underside and/or limbs), scabs on one or more areas of the body, an abrasion (head, neck, mouth or right forelimb), misaligned/missing/broken/overgrown incisors, localised alopecia (head, neck and/or limbs), red substance in the cage, discharge (clear, slight clear, slight clear red or red), urine-stained abdominal fur, chromorhinorrhea, chromodacryorrhea, perioral substance (dried brown or reddish-brown, red or red and yellow), ulceration (mouth, neck and/or right side of neck), red substance on the cage liner, swollen snout, rales, gasping, soft or liquid faeces, red substance on the penis, broken nail, hole in the palate, red urine, bent snout, swollen right ear, limited use of the right forelimb, vocalization, no use of the
right forelimb, tachypnea, scant faeces, mild or moderate dehydration, decreased motor activity, splayed forelimbs and hindlimbs, open-mouth breathing, laboured breathing and no faeces in the cage pan. Fewer P generation male rats (significant at p ≤ 0.01) in the citronellyl nitrile-treated groups had dyspnea, in comparison to the vehicle control group value.

MALE BODY WEIGHTS AND BODY WEIGHT CHANGES
Body weight gains for P generation male rats were transiently, but significantly reduced (p ≤ 0.05) in the 200 and 500 mg/kg/day dosage groups during the first week of the dosage period (DSs 1 to 8), in comparison to the vehicle control group value. Body weight gains in the 75, 200 and 500 mg/kg/day dosage groups were 95 %, 91 % and 93 % of the vehicle control group value, respectively, on DSs 1 to 8. These reductions were not dosage-dependent. Thereafter, body weight gains at 200 mg/kg/day were generally comparable to the vehicle control group values during the remainder of the dosage period. At 500 mg/kg/day, non-significant reductions in body weight gains occurred on DSs 15 to 22 (85 % of controls), DSs 36 to 43 (86 % of controls), DSs 78 to 84 (77 % of controls), DSs 99 to 106 (68 % of controls), DSs 106 to 113 (81 % of controls) and DSs 113 to 120 (84% of controls). In addition, body weight gains were significantly reduced (p ≤ 0.01; 65 % of controls) in this same dosage group on DSs 120 to 127, in comparison to the vehicle control group value. These sporadic reductions prior to cohabitation, and persistent reductions after cohabitation likely contributed to the overall significant reduction (p ≤ 0.01; 90 % of controls) in body weight gains that occurred at 500 mg/kg/day for the cumulative dosage period (DSs 1 to 127).

Body weights and body weight gains were unaffected by dosages of citronellyl nitrile as high as 200 mg/kg/day. Body weights on DS 127 (the last day of treatment for all male rats prior to euthanasia) were 94 %, 97 % and 94 % of the vehicle control group value in the 75, 200 and 500 mg/kg/day dosage groups, respectively.

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 citronellyl nitrile because the increase was transient and did not affect the overall body weight gain.

MALE ABSOLUTE AND RELATIVE FEED CONSUMPTION
- Absolute and relative feed consumption values were unaffected by dosages of citronellyl nitrile 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 citronellyl nitrile because: 1) the increases were transient; and 2) there was no corresponding change in absolute feed consumption in this dosage group.

MALE MATING AND FERTILITY
All mating and fertility parameters [numbers of days in cohabitation, rats that mated, the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during the first or second weeks of cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. All values were comparable among the four dosage groups and did not significantly differ from the vehicle control group values.

MALE NECROPSY
- There were no test substance-related necropsy observations. All necropsy observations were considered unrelated to citronellyl nitrile 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 euthanised prior to scheduled euthanasia. Gross lesions in rats that died early were previously described. 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 in the lungs.

MALE TERMINAL BODY WEIGHTS, ORGAN WEIGHTS AND THEIR RATIOS
- Terminal body weights for P generation male rats treated with 500 mg/kg/day citronellyl nitrile 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).
- Organs with statistically significant potentially citronellyl nitrile-related weight changes included the brain, liver, kidneys and spleen:
- 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. In addition, 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. There were no microscopic changes in the P generation male rats that could be correlated with the differences in brain, liver, kidney or splenic weights.

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 citronellyl nitrile 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 citronellyl nitrile because: 1) the changes were independent of dose; and/or 2) the changes were minimal and reflected normal biologic variation.

SPERM EVALUATIONS
All sperm parameters evaluated were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. Values for number and percent motile sperm, number of nonmotile sperm and total sperm count from the vas deferens and cauda epididymal sperm count and density were comparable among the four dosage groups and did not significantly differ from the vehicle control group values

FEMALE MORTALITY
- All P generation female rats survived to scheduled euthanasia

FEMALE CLINICAL OBSERVATIONS
Similar to male rats, the number of P generation female rats with excess salivation (slight and/or moderate) was significantly increased (p ≤ 0.01) in the 500 mg/kg/day dosage group, in comparison to the vehicle control group value. These increases were observed during the precohabitation, gestation and lactation periods. Slight excess salivation was observed as early as DS 10 and persisted into the lactation period, while the first occurrence of moderate excess salivation was on DS 1.

All other clinical observations during the precohabitation, gestation and lactation periods were considered unrelated to citronellyl nitrile 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. These clinical observations included a scab on the tail, ungroomed coat, chromodacryorrhea, misaligned/missing/broken incisors, red and/or yellow perioral substance, rales, urine-stained abdominal fur, sparse hair coat (limbs and/or underside), localized alopecia on the limbs and a mass on the lower midline.

The statistically significant increase (p ≤ 0.01) in the incidence of soft and liquid faeces that occurred in the 200 mg/kg/day dosage group during lactation was considered unrelated to citronellyl nitrile because the increase was independent of dose.

FEMALE BODY WEIGHTS AND BODY WEIGHT CHANGES
- Body weights and body weight gains during the precohabitation, gestation and lactation periods were unaffected by dosages of citronellyl nitrile 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 (DLs 1 to 5) and at 500 mg/kg/day on DLs 1 to 22, in comparison to the vehicle control group values. In addition, the average maternal body weight on DL 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 with citronellyl nitrile 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 DLs 1 to 5. At 75 mg/kg/day, body weight gains were significantly reduced (p ≤ 0.05) on DLs 8 to 11, in comparison to the vehicle control group value. This reduction was considered unrelated to citronellyl nitrile because the reduction was transient and independent of dose.

FEMALE ABSOLUTE AND RELATIVE FEED CONSUMPTION
Absolute and relative feed consumption values during the precohabitation, gestation and lactation periods were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. All values were comparable among the four dosage groups.

Absolute and relative feed consumption values were significantly increased (p≤0.05) in the 500 mg/kg/day dosage group on DGs 0 to 21 and at 75 and 500 mg/kg/day on DLs 1 to 5, in comparison to the vehicle control group values. These increases in feed consumption were considered unrelated to treatment with citronellyl nitrile because: 1) the increase was independent of dose; and 2) the increases were transient.

OESTROUS CYCLING, MATING AND FERTILITY
- The number of oestrous stages per 14 days was comparable among the four dosage groups during the precohabitation period.
- All mating and fertility parameters [numbers of days in cohabitation, rats that mated, the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during the first or second week of cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. All values were comparable among the four dosage groups and did not significantly differ.

NATURAL DELIVERY OBSERVATIONS
- Pregnancy occurred in 24, 22, 25 and 23 of the 25 mated P generation female rats in the 0 (Vehicle), 75, 200 and 500 mg/kg/day dosage groups, respectively. All pregnant dams delivered litters.
- Natural delivery and litter observations were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. Values for the numbers of dams delivering litters, the duration of gestation, averages for implantation sites per delivered litter, the gestation index (number of dams with one or more liveborn pups/number of pregnant rats), the numbers of dams with stillborn pups and of dams with all pups dying, litter sizes, surviving pups per litter, percent male pups per number of pups sexed per litter, live litter size at weighing and pup weight per litter were comparable among the four dosage groups.

The statistically significant increase (p ≤ 0.01) in the number of pups with an unknown vital status and the significant increase in the number that died between days 16 and 22 postpartum that occurred in the 200 mg/kg/day dosage group was considered unrelated to maternal treatment with citronellyl nitrile because these increases were independent of dose. In addition, the significant reduction (p ≤ 0.05) in the viability index that occurred at 500 mg/kg/day was not considered to be adverse because: 1) the value (94.7 %) was within the historical range of the Testing Facilitya (mean: 97.4 %; range: 93.8 % to 100 %); and 2) 5 of the 13 deaths that occurred in the 500 mg/kg/day dosage group between days 2 and 5 postpartum were in one litter (no. 16281). The lactation index was significantly increased (p ≤ 0.05 or p ≤ 0.01) in the 75 and 500 mg/kg/day dosage groups, in comparison to the vehicle control group value. These increases were considered unrelated to treatment with citronellyl nitrile because: 1) the increase was independent of dose; and/or 2) the value was within the historical range of the Testing Facility.

FEMALE NECROPSY
There were no test substance-related necropsy observations. All gross lesions were considered unrelated to treatment with citronellyl nitrile 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.

FEMALE BODY WEIGHTS, ORGAN WEIGHTS AND THEIR RATIOS
- Terminal body weights for P generation female rats treated with 500 mg/kg/day citronellyl nitrile were significantly increased (p ≤ 0.01), in comparison to the vehicle control group value.
- Organs with statistically significant potentially citronellyl nitrile-related weight changes included the liver and kidneys:
-- 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. In addition, 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 citronellyl nitrile as high as 500 mg/kg/day.
-- All other statistically significant changes were considered unrelated to citronellyl nitrile because the changes were independent of dose

HISTOPATHOLOGY
- 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 citronellyl nitrile under the conditions of this study.

OVARIAN FOLLICLE COUNTS
- The ovarian primordial follicle counts did not appear to be affected by treatment with citronellyl nitrile at any dosage level tested.
- Overall, a slight increase was noted for the mean number of primordial follicles in the 500 mg/kg/day, as compared to the vehicle control group (159 and 132 follicles, respectively). This increase was associated with higher variability than noted for the vehicle control group rats. As well, two rats in the 500 mg/kg/day dosage group (nos. 16289 and 16291) showed comparatively high numbers of primordial follicles (451 and 359 total primordial follicles per rat, respectively), most likely influencing both the noted increase from the vehicle control group rats, and the variability as well. Corpora lutea were present for all animals.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

PREWEANING: CLINICAL SIGNS
There were no treatment-related clinical signs observed in the F1 generation pups following treatment of P generation rats with citronellyl nitrile at dosages as high as 500 mg/kg/day. All transient and persistent clinical observations were considered unrelated to maternal treatment with citronellyl nitrile because: 1) the incidences were not dosage-dependent; 2) several of the observations were presumed related to the tattooing process that occurred on day 1 postpartum; and/or 3) the number of litters affected did not differ significantly from the vehicle control group values.

Transient clinical observations that occurred and were associated with poor maternal care included dehydration (mild to severe), not nursing, cold to touch, discolored head or tail (presumed bruising) and ungroomed coat. Decreased incidences (significant at p ≤ 0.01) of an absence milkband were noted in the 75, 200 and 500 mg/kg/day dosage groups in comparison to the vehicle control group value. This statistically significant change was not attributed to the test substance, but reflected an overall increase in an absent milkband in the vehicle control group. Scabbing or ulcer formation, vocalization, labored breathing, abdominal distention, red perianal substance, malformed digits, pale appearance and/or gasping occurred at low incidences in each dosage group, including the vehicle control. Observations presumed related to a tattoo injury included swollen and/or necrotic hindlimbs or hindpaws, limited or no use of hindlimb, impaired or no gripping reflex and discolored hindlimb or hindpaw.

Persistent clinical observations that occurred in the F1 generation pups included an umbilical hernia, missing tail or tip of tail, missing digit and a short digit. No other clinical observations occurred.

PREWEANING: ANOGENITAL DISTANCE AND NIPPLE ERUPTION
- Anogenital distance on days 1 or 22 postpartum in F1 male and female pups was not affected by treatment of P generation rats with citronellyl nitrile at any dosage level tested.
- Nipple eruption did not occur in any male pup at any dosage level tested. All female pups had nipples present on day 12 postpartum, with the exception of 2 from litter 16228 (F1 generation; 75 mg/kg/day dosage group).

PREWEANING: NECROPSY
There were no gross lesions observed in the F1 generation pups that survived to scheduled necropsy on day 22 postpartum. In the pups that were stillborn, found dead or humanely euthanised, no milk was present in the stomach of 5, 1, 1 and 1 F1 generation pups in the 0 (Vehicle), 75, 200 and 500 mg/kg/day dosage groups, respectively.

POSTWEANING: MORTALITY
- All F1 generation rats selected for continued evaluation postweaning survived until scheduled euthanasia.

POSTWEANING: CLINICAL SIGNS
All clinical observations that occurred in male or female rats during the postweaning period were considered unrelated to treatment of P generation rats with citronellyl nitrile because: 1) the incidences were not dosage-dependent; 2) the observations occurred in only one rat in any dosage group; and/or 3) the observations occurred only in the vehicle control group. These clinical observations included a scab, abrasion and/or ulceration on one or more areas of the body, soft or liquid faeces, chromodacryorrhea, abdominal distention, short digits on the left hindpaw, thin or sparse hair coat, localised alopecia (neck), bent tail, misaligned/missing/broken incisors, mild dehydration (based on skin turgor), red substance on the fur, ungroomed coat and a swollen snout.

POSTWEANING:BODY WEIGHT
- Body weights and body weight gains of the F1 generation male and female rats during the postweaning period were unaffected by treatment of P generation rats with citronellyl nitrile at dosages as high as 500 mg/kg/day.
- Body weight gains in F1 male rats in the 75, 200 and 500 mg/kg/day dosage groups were 106 %, 101 % and 103% of the vehicle control group value, respectively, on days 23 to 57 postpartum. Body weight gains in F1 female rats in the 75, 200 and 500 mg/kg/day dosage groups were 102 %, 107 % and 104 % of the vehicle control group value, respectively, on days 23 to 57 postpartum.
- The statistically significant increase (p≤0.05 or p≤0.01) in body weight gains that occurred in F1 generation female rats at 75 and 200 mg/kg/day on days 51 to 57 postpartum was not attributed to maternal treatment with citronellyl nitrile because: 1) the increase was transient; and 2) the increase was independent of dose.

POSTWEANING: FEED CONSUMPTION
- Absolute and relative feed consumption values for F1 generation male and female rats during the postweaning period were unaffected by treatment of P generation rats with citronellyl nitrile at dosages as high as 500 mg/kg/day.
- Absolute feed consumption values for F1 male rats in the 75, 200 and 500 mg/kg/day dosage groups were 103 %, 100 % and 101 % of the vehicle control group value, respectively, on days 23 to 57 postpartum. Absolute feed consumption values for F1 female rats in the 75, 200 and 500 mg/kg/day dosage groups were 101 %, 102 % and 104 % of the vehicle control group value, respectively, on days 23 to 57 postpartum.
- The statistically significant reductions (p ≤ 0.05) in relative feed consumption that occurred in female rats at 75 mg/kg/day (days 23 to 30 postpartum) and in male rats at 200 mg/kg/day (day 37 to 44 postpartum) were not attributed to maternal treatment with citronellyl nitrile because the reductions were transient and independent of dose.

POSTWEANING: SEXUAL MATURATION
- Sexual maturation in F1 rats was unaffected by treatment of P generation rats with citronellyl nitrile at dosages as high as 500 mg/kg/day.
- At 75 and 500 mg/kg/day, preputial separation was achieved in F1 male rats earlier (significant at p ≤ 0.05 or p ≤ 0.01) as compared to the vehicle control group value. However, the average day of preputial separation (44.6 and 45.4 days at 75 and 500 mg/kg/day, respectively) was within the historical range of the Testing Facility (mean: 46.2 days; range: 41.3 to 49.7 days), and therefore, was not considered an adverse effect of exposure (in utero or via the milk) to citronellyl nitrile.
-The average day on which vaginal opening was observed in F1 female rats was comparable among the dosage groups. In addition, the average body weight on the day sexual maturation was achieved in F1 male and female rats was comparable among the dosage groups and did not differ significantly from the vehicle control group values.

POSTWEANING: NECROPSY
- There were no test substance-related necropsy observations. All necropsy observations were considered unrelated to treatment of P generation rats with citronellyl nitrile because: 1) the incidences were not dosage-dependent; and/or 2) the observations occurred in only one rat in any dosage group. In F1 male rats, these necropsy observations included numerous red areas on the thymus or left lateral lobe of the lung, a mottled (red and dark red) appearance to the right diaphragmatic lobe of the lung, an extra lobe present on the left lateral lobe of the liver, an enlarged right testis, slight dilation of the pelvis in the right kidney, a pale appearance to the left kidney and a clear fluid-filled cyst on the left kidney. Gross lesions observed in F1 female rats included numerous red areas on the thymus, a dark red area on the right diaphragmatic lobe of the lungs and slight dilation of the pelvis in the right kidney. No other gross lesions occurred.

POSTWEANING TERMINAL BODY WEIGHTS, ORGAN WEIGHTS AND THEIR RATIOS
- There were no apparent effects of treatment of P generation rats with citronellyl nitrile on the reproductive and nonreproductive tissue weights of the F1 male or female rats at any dosage level tested.
- A slight, but statistically significant increase (p ≤ 0.01) in the absolute weight of the left epididymis occurred in F1 male rats at 500 mg/kg/day, in comparison to the vehicle control group value. This increase was considered unrelated to treatment of P generation rats with citronellyl nitrile because there was no statistically significant change in the contralateral organ or in the relative weight of the left epididymis. In addition, the statistically significant reduction (p ≤ 0.01) in the relative (% terminal body weight) weight of the left kidney for F1 male rats at 75 mg/kg/day was not attributed to exposure to citronellyl nitrile because: 1) the reduction was independent of dose; and 2) there was no statistically significant change in the contralateral organ.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Dose formulation analysis

Group  Nominal concentration (mg/mL) Mean measured concentration (mg/mL) Mean bias (%) Homogeneity (% relative standard deviation)
Start of study I 0 0 n/a n/a 
II 18.75 19.53 4.2 0.1
III 50  50.15 0.3 0.3
IV 125 125.5 0.4 0.7
End of study I 0 0 n/a n/a 
II 18.75 23.86 27.3 n/a
III 50 60.15 20.3 n/a
IV 125 150 20.0 n/a

Full results

For full results please refer to the attached supporting materials.

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the NOAEL for toxicity of citronellyl nitrile is 200 mg/kg/day.
The reproductive NOAEL in the P generation rats and the NOAEL for viability and growth of the F1 generation offspring is greater than 500 mg/kg/day.