Cyclopentanone

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 05 sep 1984 to 19 mar 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well described and GLP, but performed on an analogue

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexanone

EC Number:

203-631-1

EC Name:

Cyclohexanone

Cas Number:

108-94-1

Molecular formula:

C6H10O

IUPAC Name:

cyclohexanone

Details on test material:

Test material identity: Cyclohexanone from Allied Fiber and Plastics Company (CAS 108-94-1)
Details on test material:
* Name of test material: cyclohexanone
* Molecular formula: C6H10O2
* Molecular weight: 98.14 g
* physical state: liquid
* Substance type: pure active substance
* Analytical purity: 99.9+%
* Impurities: 0.01% water, 0.02% cyclohexanol, 0.058% esters (as cyclohexylformate), 0.003% acidity (as formic acid)
* Purity test date: data not available
* Lot/batch No: 403102 and 506241
* Expiration date of the lot/batch: data not available
* Stability under test conditions: data not available
* Storage condition of test material: stored under nitrogen

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Species: rat
- Strain: RAT STRAINS: Sprague-Dawley
- Sex: male/female
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: (P) 40 days of age ; (F1) were 29-43 days of age at the initiation of the F1 generation
- Weight at study initiation: (P) Males: ca. 156 +/- 12 g; Females: ca. 130 +/- 8 g; (F1) Males: ca. 55-63 g; Females: ca. 50-56 g
- Fasting period before study: data not available
- 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.
- Diet: certified Purina Rodent Chow #5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 25.5°C
- Humidity: 30 - 70%
- Air changes: at least 12 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

- Generation of test atmosphere / Chamber description:
Airflow rates through the flasks were 80-100 L/minute and column and flask temperatures were maintained below 105°C (flask, column and chamber temperatures were recorded hourly). Flask air flow, column and flask temperature and FMI pump rate were adjusted to achieve the target concentrations. The pressure drop was measured by minihelic gauge that calibrated against a mass flowmeter. Chamber airflow was recorded hourly.

- Test atmosphere:
* Brief description of analytical method used: gas chromatography (detector: photoionization)
* Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: monogamous cohabitation, whenever possible, was used
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal plug or sperm-positive results of vaginal smears referred to as day 0 of pregnancy
- After 5 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

(P) Males: 5 days/weeks before mating (at least 10 weeks), 5 days/week during mating (15-day maximum duration) and until initiation of the F1a weanings
(P) Females: 5-7 days/weeks before mating (at least 7+3 weeks), 7 days/weeks during mating (15-day maximum duration), 7 days/weeks during resulting pregnancies, 7 days/weeks through weaning of their F1 offspring.
(F1) Males: 5 days/weeks before mating (at least 15 weeks), 5 days/week during mating (15-day maximum duration) and until sacrifice
(F1) Females: 5-7 days/weeks before mating (at least 12+3 weeks), 7 days/weeks during mating (15-day maximum duration) and until sacrifice
Duration of test: 2 generations

Frequency of treatment:

6 hours per day, 5 days per week (for males and females pre-mating) or 7 days per week (for females only 3 weeks prior to the mating trial, during gestation days 0-20 and on lactation days 5-28).

Details on study schedule:

no data

Doses / concentrations

Remarks:

Doses / Concentrations:
F0: 250, 500 or 1000 ppm (1.0, 2.0 or 4.0 mg/L).|F1: 250, 500 or 1400 ppm (1.0, 2.0 or 5.6 mg/L).
Basis:
other: daily time-weighed average concentrations

No. of animals per sex per dose:

30 males and 30 females in the F0 and the F1a generation

Control animals:

other: conditioned air only

Details on study design:

no data

Positive control:

no

Examinations

Parental animals: Observations and examinations:

* Cage side observations: Yes
- Time schedule: one a week (each animal was removed from its cage and thoroughly examined
* Detailed clinical observations: Yes
- Time schedule for examinations of morbidity and mortality: at least twice each day
* Body weight: Yes
- Time schedule for examinations: once a week

- Estrous cyclicity (Parental animals): no
- Sperm parameters (Parental animals): no

Litter observations:

- Standardisation of litters:
* Performed on day 4 postpartum: yes
* If yes, maximum of 8 pups/litter (4/sex/litter); excess pups were killed and discarded.
- Parameters examined: the sexes and numbers of pups delivered, delivered viable, delivered stillborn, and found partially cannibalized were recorded for each litter of progeny on the day of parturition. The number of pups surviving to lactation days 1, 4, 7, 14 and 28 were determined. Individual pup weights and sexes were determined on lactation days 0, 4, 7, 14, 21 and 28 for all surviving progeny. Each 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. Assessment for neurotoxicological effects were conducted on 1 pup from each F1a litter delivered. Ophtalmologic examination was performed on all F1a progeny and all F2a and F2b weanlings
- Gross examination of dead pups: data not available

Postmortem examinations (parental animals):

- Sacrifice:
* Male animals: All surviving animals
* Maternal animals: All surviving animals
- Gross necropsy: Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Histopathology / organ weights: The tissues indicated in Table 1 (attached document) were prepared for microscopic examination and weighed, respectively

Statistics:

Quantitative continuous variables, i.e. body weights, food consumption, were analyzed by Analysis of Variance with significant differences described by that treatment further studied by multiple comparison. 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 analyzed using Chi-square analysis and Fisher's exact test.
All statistical analyses were interpreted using the untreated control group for comparison. Differences were considered significant at the p<0.05 and p<0.01 confidence levels.

Reproductive indices:

Mating index = [ Number of copulations X 100 ] / [ Number of estrus cycles* utilized ]
Fertility index = [ Number of pregnancies X 100 ] / [ Number of copulation ]
Gestation index = [ Number of parturitions X 100 ] / [ Number of pregnancies ]
Female fertility index = [ Number of pregnancies X 100 ] / [ Number of female mated ]
Male fertility index = [ Number of sires X 100 ] / [ Number of males mated ]
* Five days equals 1 estrus cycle

Offspring viability indices:

Born viable = [ No. pups delivered alive X 100 ] / [ Total No. pup delivered ]
Born dead = [No. stillbirths X 100 ] / [ Total No. pup delivered ]
Born and cannibalized = [ No. pups found partially cannibalized X 100 ] / [Total No. pup delivered ]
1 day = [ No. pups viable at lactation day 1 X 100 ] / [ No. pups born alive ]
4 day = [ No. pups viable at lactation day 4 X 100 ] / [ No. pups born alive ]
7 day = [ No. pups viable at lactation day 7 X 100 ] / [ No. pups retained at lactation day 4 ]
14 day = [ No. pups viable at lactation day 14 X 100 ] / [ No. pups retained at lactation day 4 ]
21 day = [ No. pups viable at lactation day 21 X 100 ] / [ No. pups retained at lactation day 4 ]
28 day = [ No. pups viable at lactation day 28 X 100 ] / [ No. pups retained at lactation day 4 ]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

- CLINICAL SIGNS AND MORTALITY:
No deaths were noted for treated F0 parental animals.
In F0 parental animals, following the first 2 exposures to 1000 ppm, lacrimation, ataxia and irregular breathing were noted.


- BODY WEIGHT AND FOOD CONSUMPTION:
Body weight data of F0 treated animals were comparable to the untreated control animals.

- REPRODUCTIVE PERFORMANCE:
In F0 parental animals, statistical analyses of the mating indices calculated for the treated animals revealed no significant differences.
See table 2 in the attached document.

- OTHER FINDINGS:
Urinalysis was performed in 5 randomly selected F0 parental females from each treatment level post-lactation. The overnight urine volumes collected from these females revealed an increase in the volume of urine from the 1000 ppm females ; however, no qualitative differences were noted that corresponded with this increase. All other urinalysis parameters obtained were comparable to the untreated control females.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other:

Remarks:

Clinical signs were observed in P0 animals at 1000 ppm

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Details on results (P1)

Clinical signs:
In F1 parental animals, irregular breathing, urine soaked fur, prostration, lacrimation and ataxia were the predominant observation noted post-exposure to 1400 ppm. During week 16, these animals appeared to acclimate to treatment with lethargy being the predominant post-exposure observation. Pre-exposure, 27/60 of the 1400 ppm animals had yellow/brown stained fur, and 2 males exhibited a staggering gait during week 30.

Body weight:
in F1 parental animals, the body weight data revealed significant reductions for the 1400 ppm males up the the 33th week and for the 500 ppm males only during the first week, when compared to the untreated control males.

Effect levels (P1)

Dose descriptor:

other: NOAEL Parental F1

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Remarks on result:

other:

Remarks:

Clinical signs and body weight reduction were seen at 1400 ppm

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

- VIABILITY:
During the 1st generation, F1a litter, progeny delivery and population data were similar for treated groups and the untreated control group. Progeny survival during the F1a litters was not altered by maternal exposure to cyclohexanone.
See table 3 in the attached document.

- BODY WEIGHT:
Body weight data obtained for F1a progeny were not considered to be affected by maternal cyclohexanone exposure.

- GROSS PATHOLOGY:
The examinations for progeny development generally revealed findings/anomalies which were sporadic in occurrence and which have been seen among control progeny. During the F1a litter, 1 pup from the 500 ppm groups and 1 pup from the 1000 ppm group exhibited eye opacity.
Examination of the specified tissues of the nervous system of untreated and 1000 ppm F1a progeny selected for neurotoxicologic testing did not reveal lesions in any of the tissues.

- HISTOPATHOLOGY:
Microscopic examination of the eyes from the F1a progeny revealed lenticular vacuolation for 2/115 of the 500 ppm progeny and 3/111 of the 1000 ppm progeny. The examining pathologist concluded that based on the low incidence and minimal nature of these changes, they were not treatment-related.

- OTHER FINDINGS:
Evaluation of behavioural/neurotoxicologic development of selected F1a progeny revealed no consistent statistical differences between treated and control groups. On lactation day 15, 31-56% fewer test progeny had open eyelids than the untreated control progeny ; however no dose-response pattern was apparent.
The ophthalmologist's interpretation of the examinations of the eyes 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 examined.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: No effect

Remarks on result:

other:

Remarks:

The NOAEL is higher than 1000 ppm. No effect was noted.

Results: F2 generation

Details on results (F2)

The F2a and F2b reproductive indices revealed no statistical depression for the test groups when compared to the untreated control group. However the 1400 ppm males fertility index was significantly reduced when compared to the untreated control, 250 and 500 ppm males. Additionally, mating indices for the 1400 ppm group was significantly less than the 250 ppm group during F2a and F2b mating trials.

Statistical analysis of the F2a and F2b progeny data revealed significant reductions for the mean number of 1400 ppm progeny viable during the lactation period. Progeny delivery and population data for the 250 and 500 ppm groups were comparable to the untreated control group. The % of 1400 ppm F2a and F2b progeny delivered viable and surviving to lactation days 1 and 4 (but not at lactation days 14, 21 and 28) were significantly less than seen for the untreated control. Progeny survival indices calculated for the 250 and 500 ppm groups during the F2a and F2b litters were similar to the untreated control group.

Statistical analysis of the 1400 ppm F2a and F2b progeny body weights revealed significant reduction when compared to the untreated control progeny. Because the statistical weight differences noted for the 250 and 500 ppm F2a progeny were minimal (5 to 17%) 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.
See table 4 in table 4 in the attached document.

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

500 ppm

Sex:

not specified

Basis for effect level:

viability

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Gross necropsy and tissue preparation of parent (checked (X) collected (column C), weighed (column W) and examined for histopathology (column H with X = all groups, # = control and top dose animals)

F0 parental				F1 parental
C	W	H	SYSTEM	C	W	H	SYSTEM
			digestive				digestive
			Liver	X		#	Liver
			Stomach				Stomach
			reticulo- endothelial hematopoietic				reticulo- endothelial hematopoietic
			Bone marrow^§				Bone marrow^§
			Lymph nodes				Lymph nodes
			Peyer’s patch				Peyer’s patch
			Spleen				Spleen
			Thymus				Thymus
			urogenital				urogenital
X		#	Epididymis	X		#	Epididymis
			Kidneys	X		#	Kidneys
X		#	Ovaries	X		#	Ovaries
			Primordial follicles, growing follicles, and large Corpora lutea of lactation from the post-lactation ovary				Primordial follicles, growing follicles, and large Corpora lutea of lactation from the post-lactation ovary
X		#	Prostate	X		#	Prostate
X		#	Seminal Vesicle with Coagulating Gland (if present)	X		#	Seminal Vesicle with Coagulating Gland (if present)
X		#	Testes	X		#	Testes
X		#	Uterus (with oviducts)	X		#	Uterus (with oviducts)
X		#	Vagina	X		#	Vagina
			neurologic				neurologic
			Brain	X		#	Brain
			Peripheral nerve tissue				Peripheral nerve tissue
			Spinal cord (at least 2 different locations)				Spinal cord (at least 2 different locations)
			glandular				glandular
			Adrenal glands				Adrenal glands
			Pituitary gland				Pituitary gland
			other				other
			Target organ:	X		#	Eyes

Applicant's summary and conclusion

Executive summary:

A reproduction study was conducted to ascertain the potential effects of inhalation exposure to cyclohexanone vapor upon reproductive performance, growth, and development of 2 consecutive generations of CD Sprague-Dawley albino rats. Groups of 30 males/30 females were exposed to either 0, 250, 500 or 1000 ppm during the 1st (F0) generation. 30 males/30 females were selected from the F1a litters of each group to continue on test as second (F1) generation animals. The F1 generation animals were exposed to 0, 250, 500 or 1400 ppm cyclohexanone. Assessment for potential neurotoxicologic/neuropathologic effects were conducted pre-weaning and post-weaning in each F1a litter.

The daily time-weighted average concentrations during the F0 generation were 0, 253.2, 499.2 and 1008.9 ppm and during F1 generation the daily time-weighted average concentrations were 0, 249.8, 496.9 abd 1387.2 ppm of cyclohexanone.

Inhalation exposure to 1000 ppm cyclohexanone through one generation and exposure to 250 or 500 ppm cyclohexanone through 2 consecutive generations did not adversely affect the growth, development, and reproductive performance of the CD Sprague-Dawley derived albino rats. Evaluation for behavioral/neurotoxicologic development of selected F1a progeny revealed no consistent differences betwwen treated groups and the control group.

Inhalation exposure of the CD rat (progeny from parental animals exposed to 1000 ppm) to 1400 ppm cyclohexanone through one generation resulted in exposure-related male body weight depressions, reduced progeny survival, and progeny body weight depressions.

Based on this study cyclohexanone induce in the F2 generation a reduction on the viability and on the body weight. However the fertility is not impacted and no developmental effect was seen.