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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 September 1984 to 19 November 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with GLP

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
Throughout the first generation of this study, all parent animals were exposed to 0, 250, 500 or 1000 ppm cyclohexanone (30 per sex per group). Thirty males and 30 females were selected from the F1a litters of each group to continue the test as second (F1) generation animals. The F1a progeny selected as potential F1 generation animals were exposed to 0, 250, 500 or 1000 ppm. After weaning of the last F1a litter, the F1 parental animals were selected and the 1000 ppm exposure level was increased to 1400 ppm; the 250 and 500 ppm levels remained unchanged. Assessments for potential neurotoxicologic/neuropathologic effects were conducted pre-weaning and post-weaning in each F1a litter.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Supplier: Allied Fiber And Plastics Company, Hopewell, Virginia, USA.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Portage, MI facilities of Charles River Breeding Laboratories, Inc.
- Diet: Purina Certified Rodent Chow #5002
- Age at study initiation: F0 Generation: 40 days; F1Generation: 29 to 43 days
- Average weight at study initiation: F0 Males: 156.6 g; F0 Females: 129.9 g; F1 Males: 50.1 g; F1 Females: 52.6 g
- Fasting period before study: No
- Housing: Animals were housed in one of 2 types of cages. Stainless steel, open mesh cage bank units, each containing 10 individual cubicles, were used during acclimation and all study phases, excluding mating, gestating, and lactating periods. Hanging, wire-bottom, galvanized steel caging was used during the mating trials. These cages, equipped with solid-bottom, stainless steel floorplates and nesting material (Bed-O-Cobs, Maumee, OB) were used during gestation and lactation periods. The floorplates and nesting material were supplied to gestating females on approximately the fifteenth day of gestation and were removed from the cages of lactating females when the progeny were approximately 7 days of age. During exposure, animals were individually housed in stainless steel cage bank units. Each cage bank unit contained 10 individual cubicles. During the study phase, when both F0 and potential F1 generation animals were treated, exposures were run with animals housed in 2 layers of cage bank units. All other exposures were run with animals housed in a single layer of 6 cage bank units. The cage bank units were rotated counter-clockwise, one cage bank per week.- Diet: ad libitum- Water: Filtered tap water was provided ad libitum via demand operated valves- Acclimation period: F0 animals were acclimated for 19 days

ENVIRONMENTAL CONDITIONS
- Temperature: 68 to 78 °F
- Humidity: 30 to 70%
- Photoperiod: A 12-hour light/dark cycle was maintained

IN-LIFE DATES
From: 05 September 1984
To: The last parental sacrifice occurred on 18 October 1985. Males used for the post-exposure fertility assessment study were sacrificed on 19 November 1985.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: conditioned air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The cyclohexanone inhalation exposures were conducted in 8 m³ stainless steel and glass chambers.
- Source and rate of air: Each chamber was supplied with conditioned air (HEPA and charcoal filtered) operated dynamically under a slight (0.3 in. H2O) negative pressure to prevent contamination of the surrounding area.
- System of generating test material atmosphere: The generation system for each exposure chamber consisted of an air flow meter, glass column (30 cm) with glass beads (4 mm) and heat tape, thermometer, round bottomed flask with heating mantle, glass vapour delivery tube with heat tape, Teflon delivery tube, FMI pump, test material reservoir and vent.Column and flask temperatures were maintained below 105 °C (flask, column and chamber temperatures were recorded hourly). Flask airflow, column and flask temperatures and FMI pump rate were adjusted to achieve the target concentrations. Exposures started when the test material reached the top of the heated glass bead column and ended when test material flow to the column was stopped. No accumulation of test material occurred in the flasks. All chambers were operated for at least one-half hour after the test material flow ceased. Chamber airflows were proportional to the pressure drop across an orifice placed in the chamber exhaust line. The pressure drop was measured by a minihelic® gauge that was calibrated against a mass flowmeter.
- Temperature, humidity, pressure in air chamber: Conditioned air 67-77 °F, humidity 30-70%. Chamber supply air temperature and humidity were determined hourly in the untreated control chamber with a Taylor 5522 hygrometer.
- Air flow rate: Airflow rates through the flasks were 80-100 litres per minute. Chamber airflows were recorded hourly.
- Air change rate: Airflow rates sufficient for at least 12 air changes per hour

TEST ATMOSPHERE
- Samples taken from breathing zone: Yes
- Brief description of analytical method used: Test material AnalysisThe concentration of the test material in the breathing zone within each chamber was analysed hourly using “scrub samples” with the trapping liquid being 20 mL of denatured ethyl alcohol. The chamber atmosphere was pulled through the scrubber with a vacuum pump at a rate of 1 to 2 litres per minute for 5 minutes. All exposure levels were thoroughly checked for scrubber “break through” prior to initiation of the study and it was concluded a single scrub sample was adequate. The scrub samples were quantitatively transferred into volumetric flasks and the appropriate dilutions made with denatured ethyl alcohol. Appropriate volumes were then injected into a gas chromatograph (Hewlett Packard 5710A) operated under the following conditions:
- Column: 20 x 0.125 inch stainless steel packed with 10% UCW-982 on 80-100 WAW DMCS
- Temperatures: Detector: 160 °C; Injection Port: 200 °C; and Oven: 950 °C
- Lamp Intensity: 4
- Nitrogen Flow: approximately 30 mL/minute
- Detector: HNU PID (photoionization)
- Chamber monitoring: Samples of chamber atmospheres for analytical determinations were obtained at least hourly during each exposure. The nominal concentration for each level was determined daily by measuring the weight change of each level's reservoir and the corresponding total airflow. Nominal to analytical concentration ratios were determined daily. Once each month during the study, test material distribution within each chamber was determined by measuring concentrations at six points in each chamber at the breathing level of the animals.From the first part of the study, the presence or absence of aerosol in the exposure chambers was determined by visual inspection of a light beam directed across the chamber interior. This was done once per week during this time. During the second part of the study, a California Measurements, Inc. Piezoelectric QCM Cascade Impactor was used to monitor aerosol in the exposure chambers. Measurements with this instrument were made twice per week at designated times.
Details on mating procedure:
- M/F ratio per cage: 1:1. Monogamous cohabitation, whenever possible, was used (1 male: 1 female) with the animal pairings conducted randomly employing computer-generated male/female assignments within treatment groups- Length of cohabitation: Maximum of 15 days
- Proof of pregnancy: The observation of a copulatory plug in the vagina or sperm-positive results of vaginal smears was defined as evidence of copulation.- After successful mating each pregnant female was caged: Females for which breeding was confirmed were weighed (gestation day 0) and were housed individually, terminating their mating trial.The mating trials for the "a" litters were initiated following the pre-mating period exposure to the test material and were continued for 15 consecutive days. Following a 5-day cohabitation period, males were rotated among the unbred females in their treatment group and an additional 5 days of cohabitation was allowed. This procedure was repeated for a third 5 day male/female pairing period for females which remained unbred.Following evaluation of the F2a litter data, it was decided to conduct an additional mating trial to obtain F2b litters. The mating trial for the F2b litter was initiated approximately 2 weeks after the weaning of the F2a litter and the same procedure used during the "a" litter mating trial were followed. All surviving animals were paired; however, males were not paired with females they had been exposed to during the F2a litter mating trial. In addition, sibling pairings were avoided during the F1 generation mating trials.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
2 generations. In the parent (F0) generation, animals were exposed for 10 weeks prior to the mating period. Mating was a maximum of 15 days. Males were dosed until initiation of the F1 weanlings while females were dosed until day 28 of lactation. Unbred females were dosed for 28 days. In the F1 generation, animals were exposed for 15 weeks prior to the 15 day mating period and then were dosed until sacrifice. Unbred females were dosed for 28 days post F2b mating trials.
Frequency of treatment:
The exposures were for 6 hours per day on each exposure day. Parental males were exposed 5 days per week. The parental females were exposed 5 days per week pre-mating and 7 days per week for 3 weeks prior to the mating trials. Females continued to be exposed 7 days per week during the mating trials, on gestation days 0 through 20, and on lactation days 5 through 28. Starting on gestation day 21 through lactation day 4, dams remained in the nesting cages unexposed. Females that did not conceive litters or females that did not have viable progeny were exposed 5 days a week.
Details on study schedule:
Two male and 2 female weanlings were randomly selected at weaning from each of the F1a litters, when possible, of each group to continue on test as potential F1 parental animals. After all the F1a litters were weaned, 33 males and 33 females were chosen from each treatment group population of retained progeny for F1 parental animals.In order to assess recovery of reproductive performance, ten males were selected from each treatment group of F1 generation males based on known reproductive performance during the F2b litter mating trials. All males were rested/untreated 2 days following the last exposure/treatment with the appropriate test or control material. The males were paired weekly, with 2 untreated virgin females for 4 consecutive weeks. The males were rested 1 week, paired during the sixth week of the recovery period, rested during the seventh week, and paired during week 8.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
F0 and F1 generation
Dose / conc.:
250 ppm (nominal)
Remarks:
F0 and F1 generation
Dose / conc.:
500 ppm (nominal)
Remarks:
F0 and F1 generation
Dose / conc.:
1 000 ppm (nominal)
Remarks:
F0 generation
Dose / conc.:
1 400 ppm (nominal)
Remarks:
F1 generation
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: All parental animals were assigned to treatment groups randomly by computer program. The method used by this program is documented by Carnahan, Luther, and Wilkes, Applied Numerical Methods, Wiley, 1969. The method of selection was also used for the selection of F1a litter progeny for neurotoxicological assessment and as F1 parental animals.
Positive control:
The positive control group males received a single intraperitoneal dose at 1.0 mL/kg of a 0.05% (w/v) solution of triethylenemelamine in deionised water.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice each day for mortality, morbidity and overt signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once each week each animal was removed from its cage and thoroughly examined.

BODY WEIGHT: Yes
- Time schedule for examinations: All parental animals were weighed weekly during the premating period. Weekly body weights were obtained for all surviving parental males following completion of the mating trials, for all females which did not retain a litter and for all unbred females until their sacrifice. The parental females were weighed on gestation days 0, 6, 15 and 20 and lactation days 0, 5, 7, 14, 21 and 28. Final body weights were obtained for each animal at sacrifice or death.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: During all phases of the study, food consumption was monitored visually.

GESTATION AND LACTATION
- Each female was observed daily through gestation day 18. Gravid animals were supplied with nesting material at approximately 15 days of gestation. Starting on gestation day 19, pregnant females were examined twice daily for signs of parturition. Conception was confirmed by the observation of a vascular membrane and/or the detection of progeny by palpation.The females were allowed to deliver their litters, and daily observations of the females and young were conducted throughout lactation. Litters were weaned at 28 days of age.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.Litters in excess of 8 pups were reduced to that number on lactation day 4 using computer-generated random selections. When possible, 4 males and 4 females in each litter were retained, however if less than 4 of either sex were surviving at day 4, all survivors of that sex were retained along with the appropriate number of pups of the other sex to obtain a total of 8 progeny.

PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring:
- PopulationThe sexes and numbers of pups delivered, delivered viable, delivered stillborn, and found partially cannibalised were recorded for each litter of progeny on the day of parturition. The numbers of pups surviving to lactation days 1, 4, 7, 14, 21 and 28 were determined.
- Progeny were weaned on lactation day 28. In addition, any progeny found dead during the lactation periods, along with those sacrificed on lactation day 4, were preserved, by litter, in 70% ethyl alcohol.- Body WeightsIndividual pup weights and sexes were determined on lactation days 0, 4, 7, 14, 21 and 28 for all surviving progeny.-ObservationsEach litter of progeny was examined daily for mortality and behavioural anomalies. Each pup was also examined thoroughly for developmental anomalies at birth, at each body weight interval, and again at weaning.

NEUROTOXICOLOGICAL ASSESSMENT
- Assessments for neurotoxicological effects were conducted on 1 pup from each F1a litter delivered (survival permitting). One male or female was randomly selected from alternating litters for these procedures. This selection occurred on lactation day 4 following appropriate litter reductions. 1. Suckling Young Test ProceduresTesting began at 4 days of age and continued until the physical change tested for was present. The tests included in the suckling young evaluations were incisor eruption, eye opening, pinna detachment, surface righting, air righting, and auditory startle. 2. Post Weaning Test ProceduresTen males and 10 females per treatment group (11 males and 9 females were retained from the 500 ppm group) were randomly selected from the population of F1a progeny chosen for pre-weaning neurotoxicologic testing and were evaluated at 28, 35, 42 and 49 days of age for: lacrimation; salivation; pupil size; pupil response; corneal response; visual placing; finger approach; respiration; touch escape; toe pinch; tail pinch; grasp irritability; body tone; abdominal tone; limb rotation; limb tone; body position; locomotor activity; spatial locomotion; hypotonic gait; impaired gait; pelvic elevation; tail elevation; positional passivity; righting reflex; wire manoeuvre; grip strength: startle response; vocalisation; tremors; twitches; convulsions; piloerection; hypothermia; urination-defecation; diarrhoea; and acute death.3.

OPHTHALMOLOGIC EXAMINATIONS
- The lenses of all F1a progeny with open eyelids and all surviving F2a and F2b weanlings were examined by a board certified veterinary ophthalmologist.

GROSS EXAMINATION OF DEAD PUPS: Yes
Postmortem examinations (parental animals):
TERMINAL PROCEDURES
- The F0 parental males were sacrificed at the completion of the F1a litter weanings. The F0 parental females that failed to breed were sacrificed 20 days following completion of the F1a mating trials; F0 females that bred but failed to deliver viable progeny (i.e. not gravid or resorbed) were sacrificed 26 days post copulation. Females that conceived and delivered progeny were sacrificed after completion of the F1a litters.All animals that were sacrificed or died prior to final sacrifice were necropsied. In addition, overnight urine samples were collected from 5 lactating F0 females per group, a total of 20. After the last exposure (on lactation day 28) the females were placed in urine collection cages until the following morning. They were then anaesthetised with ether and necropsied after their blood was obtained from the dorsal aorta.The volume of the overnight urine samples was measured and the urine was tested for glucose, pH, protein, ketone, bilirubin, occult blood, and urobilinogen using a dipstick procedure. The urine samples were then stored frozen at -20 °C. The serum was separated from the red cells and stored frozen at -20 °C; the cellular portion of the blood samples was discarded.

PATHOLOGY
- All F0 and F1 parental animals, sacrificed and found dead, were subjected to gross necropsy examination. With the exception of the F0 parental females that were bled and the F1 parental males that were perfused in situ, the sacrificed animals were rendered unconscious by carbon dioxide and exsanguinated. The F0 females that were bled were rendered unconscious using ether anaesthesia prior to blood collection and sacrifice.The necropsy included examination of the external body surface and all orifices; cranial cavity; external and cut surfaces of the brain and spinal cord; thoracic, abdominal and pelvic cavities and their viscera; cervical tissues and organs; and the carcass. Additionally the number or uterine implantation scars was noted and recorded for all dams.The vagina, uterus and ovaries or testes (with epididymides), seminal vesicles and prostate and any masses or gross lesions were retained in individual, labelled jars containing 10% buffered formalin. In addition, the eyes were retained from all F1 parental animals. The liver, kidney(s) (at least one or one-half of each), brain (at least one fourth), and ovarie(s) (one) or testes (one) were retained from 2 F1 parental generation males and 2 F1 parental generation females from each exposure group. These tissues were frozen using liquid nitrogen and stored at approximately -80 °C. Additionally, as a result of clinical observations noted for 2 of the 1400 ppm F1 parental generation sibling males (AG3824, AG3025), these males along with 2 males chosen randomly from the remaining 1400 ppm males and 4 of the 0 ppm males were anaesthetised and perfused in situ.Microscopic examinations were conducted upon the above listed tissues from the sacrificed untreated control and high dose parental animals from both generations.
Postmortem examinations (offspring):
TERMINAL PROCEDURES
- 1. F1a ProgenyTwo male and 2 female weanlings, when possible, were selected from each of the F1a litters weaned of each treatment group to continue on test as potential F1 parental animals. Progeny selected for neurotoxicological testing were not included in these selections. After weaning of all F1a litters, 30 males and 30 females were selected from each treatment group to serve as F1 parental animals. Those progeny not selected as F1 parental animals or for neurotoxicologic testing were sacrificed and subjected to pathologic examination.
- 2. F2a and F2b progenyAll surviving F2a and F2b weanlings were sacrificed and subjected to gross pathologic examinations.

PATHOLOGY
- All F1a progeny in excess of those chosen as F1 parental animals, or for neuropathology and all F2a and F2b progeny were sacrificed using CO2 asphyxiation, exsanguinated, and subjected to gross pathologic examination. The necropsy included an examination of the external surface; all orifices; cranial cavity; carcass; external and cut surfaces of the brain and spinal cord; the thoracic, abdominal, and pelvic cavities and their viscera; and the cervical tissues and organs. The eyes and any gross lesions were retained in 10% buffered formalin. Microscopic examinations were conducted upon the eyes of the sacrificed F1a progeny and F1a progeny chosen for neurotoxicologic testing.

NEUROPATHOLOGY
- The F1a progeny chosen for neurotoxicological assessments were sacrificed at 49 days of age, following completion of the in-life testing. These animals were anaesthetised using an intraperitoneal injection of sodium pentobarbital and perfused in situ, initially with heparinized phosphate buffered saline followed by perfusion with 10% neutral buffered formalin for fixation. The cranium and vertebral column were exposed and the animals stored in fixative at 4 °C for at least 12 hours. The cranium and vertebral column were removed without damage to brain and cord, the brain was then measured (length and width), its weight recorded, and any abnormal coloration or discoloration noted and recorded. The proximal sciatic nerve, sural nerve, tibial nerve and gastrocnemius muscle were taken. These tissues were further fixed, stored for at least 48 hours, prior to further processing.The volume of fixative versus the volume of tissues in a specimen jar was no less than 25:1. Tissues listed below retained from the control and high-dose group animals were subjected to microscopic evaluation:- Brain: forebrain, centre of cerebrum, midbrain, cerebellum and pons, medulla oblongata, and Gasserian ganglia.- Spinal Cord: At cervical (C3-C6), lumbar (L1-L4) swellings, dorsal root ganglia (C3-C6 , L1 -L4), and dorsal and ventral root fibres (C3-C6, L1 -L4).- Sciatic Nerve: Mid-thigh and sciatic notch, sural nerve (at knee), and tibial nerve (at knee).
Statistics:
Quantitative continuous variables, i.e., body weights and food consumption, were analysed by Analysis of Variance with significant differences described by that treatment further studied by multiple comparison (Tukey’s or Scheffe’s, dependent upon ‘N’ values). Progeny body weight data were additionally studied using Analysis of Covariance (with the litter size as the covariate) and Dunnett's T-test. Reproductive data and neurotoxicologic data were analysed using Chi-square analysis and Fisher's Exact test.Unless indicated otherwise, all statistical analyses were interpreted using the untreated control for comparison. Differences were considered significant at the p<0.05 and p<0.01 confidence levels.
Reproductive indices:
Mating Index = (Number of Number of copulations / Number of oestrus cycles* utilised) x 100Fertility Index = (Number of pregnancies / Number of Number of copulations) x 100Gestation Index = (Number of parturitions / Number of pregnancies) x 100Female Fertility Index = (Number of pregnancies / Number of females mated) x 100Male Fertility Index = (Number of sires / Number of males mated) x 100*Five days equals 1 oestrus cycle
Offspring viability indices:
Born Viable = (Number of pups delivered alive / Total number of pups delivered) x 100Born Dead = (Number of stillbirths / Total number of pups delivered) x 100Born and cannibalised = (Number of pups found partially cannibalised / Total number of pups delivered) x 1001 Day = (Number of pups viable at lactation day 1 / Number of pups born alive) x 1004 Day = (Number of pups viable at lactation day 4 / Number of pups born alive) x 1007 Day = (Number of pups viable at lactation day 7 / Number of pups born alive) x 10014 Day = (Number of pups viable at lactation day 14 / Number of pups born alive) x 10021 Day = (Number of pups viable at lactation day 21 / Number of pups born alive) x 10028 Day = (Number of pups viable at lactation day 28 / Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No noteworthy observations were seen for F0 animals pre-exposure. Clinical reactions, such as lacrimation, ataxia and irregular breathing, were noted for the 1000 ppm animals following the first 2 exposures. Starting with the third exposure, these animals appeared to acclimate to the test material and no consistent, recurring observations were noted post-exposure for the 1000 ppm animals through the remainder of the exposure period. No post-exposure reactions were seen for the 250 or 500 ppm animals.
Mortality:
no mortality observed
Description (incidence):
During the first generation, no deaths occurred among the treated animals. Two untreated control females died prior to final sacrifice. One dam was sacrificed moribund and one dam was found dead following completion of their respective F1a litters.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight data for the F0 parent animals exposed to the test material were comparable to the untreated control animals.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis determinations of 5 F0 females per treatment group post-lactation revealed increased volume from the 1000 ppm animals; however, no qualitative differences were noted in glucose, pH, protein, ketone, bilirubin, occult blood or urobilinogen. All other urine parameters for treated females were comparable to the untreated control females.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic changes were seen in the reproductive organs from the 1000 ppm animals and the untreated control animals.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE
- Statistical analyses of the mating indices calculated for the treated animals during the F1a mating trials revealed no significant differences. Mating indices and male fertility indices for the 500 and 1000 ppm dose groups ranged from 13 to 20 % less than the control group, but these were not statistically significant. All other parameters were comparable to the control.

PROGENY
- Progeny data (delivery and population data, survival, and body weight data) obtained during the F1a litter were not considered to be altered by maternal exposure to cyclohexanone.The observations of gestating and/or lactating dams for untoward reactions revealed no effects which were considered to be related to the treatment with the test material.

Effect levels (P0)

Key result
Dose descriptor:
NOAEC
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
no
System:
male reproductive system
Organ:
seminal vesicle
testes
other: epididymides
Key result
Critical effects observed:
no
System:
female reproductive system
Organ:
ovary
uterus
vagina

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Observations recorded prior to exposure revealed 27/60 of the 1400 ppm animals had yellow/brown stained fur in comparison to 3/60 of the untreated control group. In addition, starting at week 30 of the F1 generation and continuing through termination, two sibling 1400 ppm males exhibited a staggering gait prior to test material exposure.Exposure of F1 parental animals to 1000/1400 ppm resulted in noteworthy pharmacotoxic reactions. The F1a progeny exposed to 1000 ppm (post-weaning, prior to the selection of the F1 parental animals and subsequent increase to 1400 ppm) exhibited clinical signs such as ataxia, lacrimation, irregular breathing, and urine soaked fur following treatment. After the increase to 1400 ppm, and continuing for approximately 3 months, these reactions (along with prostration in the first week of 1400 ppm exposure) continued to occur. Starting at week 16 of the F1 generation, the 1400 ppm animals appeared to adapt to treatment with lethargy being the predominant post-exposure reaction. No observations were noted post-exposure during the final 3 weeks of the F1 generation. During the first 3 weeks of exposure, urine soaked fur was noted post-exposure for 3 to 37% of the animals exposed to 500 ppm cyclohexanone. No other noteworthy reactions were seen among the 500 ppm animals. No untoward reactions were seen for the F1 generation animals exposed to 250 ppm cyclohexanone.
Mortality:
mortality observed, treatment-related
Description (incidence):
Six of the F1 generation animals exposed to 1400 ppm cyclohexanone died. Two males and 1 female died during the first week of exposure. One male died during the fifteenth week of the pre-mating period and one male died during the F2b mating trials. One of the males used for the post-exposure assessment of fertility was found dead on the scheduled day of sacrifice. A 250 ppm male was sacrificed moribund prior to the F2b mating trials; no other deaths occurred among the 250 and 500 ppm animals or the untreated control animals during the second generation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Starting with the first week of exposure to 1400 ppm, statistically significant (p<0.01, p<0.05) weight depressions were noted for the 1400 ppm males when compared to the untreated control males. These depressions were seen at 29 of the subsequent 33 weeks of 1400 ppm exposure. Females from this exposure level weighed less (p<0.05) than the untreated control females during the week of exposure to 1000 ppm. Body weights for these females were reduced (p<0.01) at the first week of 1400 ppm exposure and these depressions (p<0.05) continued during weeks 3 and 4. Starting with the fourth week of 1400 ppm exposure through final sacrifice, no significant body weight differences were seen for the 1400 ppm females when compared to the untreated control females.During the first week of exposure, a significant weight depression (p<0.05) was seen for the 500 ppm males when compared to the untreated control males. All other body weight data obtained for the 250 and 500 ppm animals were similar to the untreated control animals. In addition, body weight data recorded for gestating and lactating dams were similar for the treated groups and the untreated control group during both generations.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Ophthalmologic examinations of the F1a progeny revealed lens opacities for 2/296 of the 250 ppm progeny (unilateral), 2/174 of the 500 ppm progeny (unilateral), and 1/174 of the 1400 ppm progeny (bilateral). The 250 and 500 ppm F1a progeny were retained, unexposed, to determine if the findings would reverse. Approximately 3 months following the initial examination, these animals were re-examined. One 250 ppm male which initially had a thread-like white opacity (unilateral) was normal at the subsequent examination; one 250 ppm male with a cloudy anterior lens capsule (unilateral) had an anterior cortical cataract at the subsequent exam; a 500 ppm male that earlier had a lens capsule which appeared cloudy (unilateral) had a roughened cornea and normal lens at the subsequent examination; and a 500 ppm male with an incipient cataract involving the nucleus of the lens (unilateral) had a granular corneal surface and nuclear opacity in lens of the previously affected eye and pinpoint opacities on the posterior lens cap of the eye that appeared normal at the initial examination. Due to the low incidence and the minimal nature of these effects, the pathologist concluded that they were not related to treatment.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Evaluation of behavioural/neurotoxicologic development of selected F1a progeny revealed no consistent statistical differences between treated and control groups. On lactation day 15, 31 to 56 percent fewer test progeny had open eyelids than the untreated control progeny; however no dose-response pattern was apparent.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathologic examinations of all F1 parental animals revealed no consistent lesions which were considered to be treatment-related.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Neuropathologic examination of tissues from the sibling males that were ataxic revealed no morphologic abnormalities. Examination of the specified areas of the nervous system of the untreated control and 1000 ppm F1a progeny chosen for neurotoxicologic evaluation did not reveal lesions in any of the tissues. Microscopic examination of the eyes from the F1a progeny revealed lenticular vacuolation (vacuolation of a few outer cortical fibres in the lens) for 2/115 of the 500 ppm progeny and 3/114 of the 1000 ppm progeny. The examining pathologist concluded that due to the low incidence and minimal nature of these changes, they were not treatment-related.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination of the reproductive organs from the untreated control and 1400 ppm parent animals revealed no evidence of treatment-related effects.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE
- Statistical analyses of the reproductive indices showed no statistical depressions for the test groups when compared to the untreated control group. However, the fertility indices for male rats exposed to 1400 ppm calculated using all males paired were 19.8 to 20.8% less than the untreated control males during the F2a and F2b litters, respectively. The male fertility calculated using only the animals that were paired with females conceiving litters showed a 24.3 to 28.6% reduction for the males exposed to 1400 ppm compared to the untreated control males during the F2a and F2b mating trials, respectively. Evaluation of these data for intergroup differences revealed significant depressions (p <0.05) for the 1400 ppm male fertility index when compared to the 250 ppm males during the F2a and F2b mating trials and compared to the 500 ppm males in the F2b mating trials. Furthermore, mating indices calculated for the 1400 ppm group were significantly less than those in the 250 ppm group during both the F2a (p <0.01) and F2b (p <0.05) mating trials.

Effect levels (P1)

Key result
Dose descriptor:
NOAEC
Effect level:
500 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance

Target system / organ toxicity (P1)

open allclose all
Key result
Critical effects observed:
no
System:
male reproductive system
Organ:
seminal vesicle
testes
other: epididymides
Key result
Critical effects observed:
no
System:
female reproductive system
Organ:
ovary
uterus
vagina

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Statistical analyses of the F2a and F2b progeny data revealed significant (p <0.01, p<0.05) reductions for the mean number of 1400 ppm progeny viable during lactation periods. Furthermore, the 1400 ppm dams delivered 23 and 24% fewer viable progeny than the untreated control dams during the F2a and F2b litters; however, no statistically significant differences to the controls were noted. Progeny delivery and population data for the 250 and 500 ppm groups were comparable to the data of the untreated control group.The percentage of 1400 ppm F2a progeny delivered viable and surviving to lactation days 1 and 4 were significantly less (p <0.01) than in the untreated control. Progeny survival of the 1400 ppm F2a progeny at lactation days 14, 21 and 28 was not statistically different from the untreated control group; the survival indices on these days were 14 to 22% less than that of the untreated control progeny. During the F2b litter, 1400 ppm progeny survival to lactation days 1 and 4 was statistically less (p <0.01) than that of the untreated control progeny. Survival of 1400 ppm F2b progeny after lactation day 4 (lactation days 7, 14, 21 and 28) was comparable to the untreated control progeny survival. Progeny survival indices calculated for the 250 and 500 ppm groups during the F2a and F2b litters were similar to the untreated control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistical analyses of the 1400 ppm F2a and F2b progeny body weights revealed significant reductions (p <0.05, p <0.01) when compared to the untreated control progeny using both the Analyses of Variance of the individual pup weights and Analyses of Covariance of the mean litter weight data. Analyses of the 250 and 500 ppm F2a progeny body weights resulted in statistical reductions (p <0.05) in individual weights at lactation days 0, 14, 21 and 28. The mean litter weights for 250 ppm progeny were reduced (p <0.05) at lactation day 21. Statistical depressions were noted for the 500 ppm mean litter weights at lactation day 21 (p <0.01) and mean male litter weights at lactation day 28 (p <0.05). The F2b progeny from these dams exhibited significant (p <0.05, p <0.01) weight increases on lactation days 0 and 4 when analysed using individual body weight data. Because the statistical weight differences noted for the 250 and 500 ppm F2a progeny were minimal (5 to 17% less than the untreated control progeny) and were not seen for the F2b progeny, maternal exposure to 250 or 500 ppm cyclohexanone was not considered to adversely affect pup body weights.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of the F2a progeny, post-weaning, revealed lens opacities or cloudiness for 3/151 of the 250 ppm progeny (unilateral) and 1/60 of the 1400 ppm progeny (unilateral). Additionally, 1/159 of the 500 ppm progeny had a corneal opacity. Ophthalmologic examination of the F2b progeny, post-weaning, for lens opacities or cloudiness revealed no finding for treated or control groups. The ophthalmologist's interpretation of the examinations was that the test substance did not increase the incidence of cataracts in the progeny. The lens and other ocular abnormalities appeared to be within the range of type and incidence expected in the number of animals expected.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy examinations of progeny revealed no lesions which appeared to be related to cyclohexanone.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
4 100 mg/m³ air (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Any other information on results incl. tables

Table 1: Reproductive Performance

Dose Group (ppm)

Mating Index (percent)

Fertility Index (percent)

Gestation Index (percent)

Female Fertility Index (percent)

Male Fertility Index (percent)

Average Gestation Length (days)

F0 Generation - F1a Litter

0

88.2

96.7

96.6

96.7 (100.0)

83.3 (86.2)

22

250

88.2

93.3

96.4

93.3 (100.0)

80.0 (85.7)

22

500

73.2

96.7

100.0

96.7 (100.0)

66.7 (69.0)

22

1000

70.7

93.1

92.6

90.0 (96.4)

70.0 (75.0)

22

F1 Generation - F2a Litter

0

46.9

91.3

95.2

70.0 (80.8)

60.0 (81.8)

22

250

71.1*

85.2

100.0

76.7 (88.5)

73.3 (91.7)

22

500

57.8

88.5

95.6

76.7 (85.2)

56.7 (73.9)

22

1400

44.4***

75.0

94.4

62.1 (72.0)

48.1** (61.9)

22

F1 Generation - F2b Litter

0

44.0

77.3

100.0

56.7 (77.3)

46.7 (82.4)

22

250

56.8

84.0

95.2

70.0 (84.0)

62.1 (90.0)

22

500

82.2

75.0

100.0

60.0 (81.8)

53.3 (88.9)

22

1400

35.7**

85.0

94.1

58.6 (70.8)

37.0† (58.8)

22

 NB: values in parentheses represent the indices omitting either females that were paired only with males that failed to sire an F1a litter or males that were only paired with females that failed to conceive an F1a litter.

*Statistically significantly different from the control group at the 95 % confidence level

**Statistically significantly different from the 250 ppm group at the 95 % confidence level

***Statistically significantly different from the 250 ppm group at the 99 % confidence level

† Statistically significantly different from the 250 and 500 ppm groups at the 95 % confidence level

 

Table 2: Total Progeny Survival

Dose Group (ppm)

Percent of Progeny Born

Progeny Survival (percent) on Lactation Day

Viable

Dead

Cannibalised

1

4

7

14

21

28

F0 Generation - F1a Litter

0

98.7

1.3

0.0

99.2

95.5

93.7

82.0

77.0

77.0

250

98.9

1.1

0.0

99.2

96.2

99.5

98.6

95.4

95.4

500

97.8

2.0

0.2

98.5

94.4

99.1

86.6

79.0

78.1

1000

100.0

0.0

0.0

95.7

93.7

100.0

97.4

92.6

92.1

F1 Generation - F2a Litter

0

96.7

3.3

0.0

99.6

97.4

98.7

93.5

92.9

92.9

250

99.3

0.4

0.4

98.9

97.1

98.8

91.3

87.9

87.9

500

95.2

4.4

0.4

95.4

92.7

99.4

99.4

98.8

98.8

1400

85.6*

12.8

1.7

77.9*

75.3*

96.4

79.5

79.5

72.3

F1 Generation - F2b Litter

0

98.1

1.9

0.0

100.0

99.0

99.2

99.2

99.2

98.5

250

99.6

0.4

0.0

94.5

94.1

99.3

94.6

94.6

94.6

500

99.5

0.5

0.0

99.0

94.9

96.9

95.3

95.3

95.3

1400

97.4

2.0

0.7

75.2*

69.1*

96.9

92.3

92.3

92.3

Data in this table reflect the progeny survival as a group

*Statistically significantly different at the 99 % confidence level

 

Table 3: Progeny Body Weight Data (g)

Dose Group (ppm)

 

Mean Values on Lactation Day

0

4

7

14

21

28

Male

Female

F0 Generation - F1a Litter

0

Mean

SD

N

5.4

0.8

372

7.8

1.5

357

10.8

2.7

208

19.0

4.5

182

31.6

7.3

171

61.6

13.1

86

55.7

11.3

85

250

Mean

SD

N

5.4

0.7

371

7.9

1.3

359

10.3

2.1

215

17.8*

3.1

213

28.2**

5.4

206

55.0**

9.8

101

52.7

9.8

105

500

Mean

SD

N

5.4

0.8

395

7.6

1.6

373

9.7**

2.5

222

16.0**

4.7

194

26.9**

7.0

177

50.9**†

14.0

87

48.8**

13.9

88

1000

Mean

SD

N

5.5

1.0

294

8.3**

1.6

283

10.0

2.3

189

17.6*

4.0

184

28.9**

5.9

175

56.5

11.7

86

53.8

10.2

88

F1 Generation - F2a Litter

0

Mean

SD

N

5.9

0.9

235

7.9

1.5

229

11.7

2.4

125

19.7

0.7

144

32.2

6.2

143

62.7

10.4

70

66.0

11.4

73

250

Mean

SD

N

5.6**

0.8

274

8.2

1.8

267

11.4

2.4

171

17.8**

3.9

158

28.4**‡

6.0

152

53.1**

13.1

78

51.0

12.2

74

500

Mean

SD

N

5.5**

0.8

259

8.1

1.6

240

11.3

2.1

161

17.9**

3.5

161

27.2**†

5.7

160

52.2**‡

12.8

78

49.5**

10.7

82

1400

Mean

SD

N

5.0**†

0.8

150

7.8

1.1

116

9.1**†

2.0

80

14.3**†

2.0

66

20.8**†

4.6

66

38.8**†

9.3

34

37.5**†

9.9

26

F1 Generation - F2b Litter

0

Mean

SD

N

5.9

0.8

209

8.9

1.4

207

13.1

1.5

129

22.5

3.7

129

35.5

6.9

129

67.8

11.0

63

64.6

9.3

65

250

Mean

SD

N

6.2*

0.9

255

9.3*

1.3

240

12.9

2.2

147

23.3

2.8

140

36.8

5.2

140

69.2

9.0

67

56.2

7.9

73

500

Mean

SD

N

6.2*

1.0

197

9.5**

1.5

187

13.5

1.8

124

22.0

3.4

122

34.6

6.5

122

69.2

11.3

59

62.1

10.9

63

1400

Mean

SD

N

5.8

1.2

142

8.3*

1.7

103

10.8**†

2.3

63

17.3**†

4.0

60

26.0**†

6.3

60

51.9**

15.7

30

47.9**†

11.5

30

SD = Standard deviation

N = Number of animals

*Statistically significantly different at the 95 % confidence level (individual progeny body weight data) using ANOVA and Scheffe’s multiple comparison

** Statistically significantly different at the 99 % confidence level (individual progeny body weight data) using ANOVA and Scheffe’s multiple comparison

‡ Statistically significantly different at the 95 % confidence level (mean litter weight data) using ANCOVA and Dunnett’s t-test

† Statistically significantly different at the 99 % confidence level (mean litter weight data) using ANCOVA and Dunnett’s t-test

Applicant's summary and conclusion

Conclusions:
Inhalation exposure to 1000 ppm cyclohexanone through one generation and exposure to 250 or 500 ppm cyclohexanone through two consecutive generations did not adversely affect the growth, development, and reproductive performance of the rat. Evaluation for behavioral/neurotoxicologic development of selected progeny revealed no consistent differences between treated groups and the control group.The NOAEC values were therefore 1000 ppm (4.01 mg/L) for the F0 generation and 500 ppm (2.01 mg/L) for the F1 and F2 generations.
Executive summary:

A reproduction toxicity study was conducted to ascertain the potential effects of inhalation exposure to cyclohexanone vapour upon growth, development, and reproductive performance of 2 consecutive generations of CD® Sprague Dawley derived albino rats. The method was broadly equivalent to that of the standardised guideline OECD 416 and the study was conducted under GLP conditions.

Groups of 30 males and 30 females were exposed by inhalation to 0, 250, 500 or 1000 ppm during the first parent (F0) generation. Thirty males and 30 females were selected from the F1a litters of each treatment group to continue on test as second parent (F1) generation animals. The F1 generation animals were exposed to 0, 250, 500 or 1400 ppm cyclohexanone (increased to 1400 ppm after 1 week of exposure to 1000 ppm). Assessments for neurotoxicologic effects were conducted pre-weaning on one pup from each F1a litter. Twenty-eight of the 0 ppm progeny, 27 of the 250 ppm progeny, 29 of the 500 ppm progeny and 25 of the 1000 ppm progeny were selected for pre-weaning testing. Post-weaning neurologic testing and neuropathologic evaluations were conducted on 20 (10 males and 10 females, survival permitting) F1a progeny per treatment group chosen from those tested pre-weaning.

There were no treatment related effects during the first generation on parental animals, reproduction, or on the F1a pups.

Six of the F1 generation animals exposed to 1400 ppm died (5 males and 1 female). In addition reduced body weight gains were noted in 1400 ppm animals during some periods. All other body weight data obtained for the 250 and 500 ppm animals were similar to the untreated control animals. Furthermore, body weight data recorded for gestating and lactating dams were similar for the treated groups and the untreated control group.

The F1a progeny exposed to 1000 ppm (post-weaning, prior to the selection of the F1 parental animals and subsequent increase to 1400 ppm) exhibited clinical signs such as ataxia, lacrimation, irregular breathing and urine soaked fur following treatment. After the increase to 1400 ppm, and continuing for approximately 3 months, these reactions continued to occur. Starting at week 16 of the F1 generation, the 1400 ppm animals appeared to adapt to treatment with lethargy being the predominant post-exposure reaction. No observations were noted post-exposure during the final 3 weeks of the F1 generation.

During the first 3 weeks of exposure, urine soaked fur was noted post-exposure for 3 to 37% of the animals exposed to 500 ppm. No other noteworthy reactions were seen among the 500 ppm animals. No untoward reactions were seen for the F1 generation animals exposed to 250 ppm.

Statistical analysis of the reproductive indices in the F2a and F2b mating trials revealed no statistical depressions for the test groups when compared to the untreated control group. However, the 1400 ppm male fertility indices, calculated using all males paired were 19.8 and 20.8 percent less than the untreated control males during the F2a and F2b litters, respectively. Also, male fertility calculated including only males which were paired with fertile females (females that conceived litters) were 24.3 to 28.6 percent less than the untreated control males during the F2a and F2b mating trials.

Statistical analyses of the progeny population data revealed significant depressions in the mean numbers of 1400 ppm viable progeny during the F2a and F2b lactation periods. The mean number of progeny born viable by 1400 ppm dams was not statistically reduced; however, in comparison to the untreated control dams, the 1400 ppm dams delivered 23 and 24% fewer viable progeny during the F2a and F2b litters, respectively. Progeny delivery and population data for the 250 and 500 ppm groups during the F2a and F2b litters were similar to the untreated control group.

The percent of 1400 ppm F2a progeny born viable and surviving to lactation days 1 and 4 were significantly less than in the untreated control group. The percentag of F2a progeny surviving during lactation up to day 21 was 14 to 22% less than that of the untreated control, although statistical significance was not achieved. During the F2b litter, progeny survival was significantly less than that of the untreated control progeny at lactation days 1 and 4. Survival of 1400 ppm F2b progeny after lactation day 4 was comparable to that of the untreated control. Survival of the 250 and 500 ppm progeny during the F2a and F2b litters was not altered by maternal exposure to cyclohexanone.

Body weights obtained for the 1400 ppm F2a and F2b progeny were depressed when compared to the untreated control progeny. No body weight reductions were noted for the 250 and 500 ppm F2a and F2b progeny which were considered to be treatment-related. Examination for progeny external morphologic changes revealed no anomalies attributable to maternal cyclohexanone exposure.

Gross and microscopic pathologic examinations of all F1 parental animals and F2a and F2b progeny revealed no treatment-related effects.

In conclusion, inhalation exposure to 1000 ppm cyclohexanone through one generation and exposure to 250 or 500 ppm cyclohexanone through two consecutive generations did not adversely affect the growth, development, and reproductive performance of the rat. Evaluation for behavioural/neurotoxicologic development of selected F1a progeny revealed no consistent differences between treated groups and the control group.

The NOAEC values were therefore 1000 ppm (4.01 mg/L) for the F0 generation and 500 ppm (2.01 mg/L) for the F1 and F2 generations.