Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
15 Jun 1995 - 10 Apr 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study conducted according to a previous guideline version. Therefore, comparable to guideline study with acceptable restrictions. No organ weights determined, except for testes; no sperm parameters and estrous cyclicity examined. Refer to endpoint summary Toxicity to reproduction and IUCLID Section 13 for reporting and justification of the analogue approach.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted 23 May 1983
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
no organ weights determined, except for testes; no sperm parameters and estrous cyclicity examined
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cyclohexane
- Molecular formula: C6H12
- Molecular weight: 84.16
- Smiles notation: C1CCCCC1
- InChl: 1S/C6H12/c1-2-4-6-5-3-1/h1-6H2
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: > 99.9% by analysis
- Purity test dates: 02 Aug 1995; 10 and 26 Nov 1995; 05, 10 and 11 Jan 1996; 10 and 11 Mar 1996; 30 Sep 1996 and 01 Oct 1996
- Supplier: Phillips Petroleum Company, Sweeney Refinery, Sweeney, Texas
- Laboratory identification number: 21174
- Stability under test conditions: stable over the duration of the study
- Storage condition of test material: in stainless steel drums
- Other: The test substance was supplied as a liquid in 55-gallon stainless steel drums. Drums identified as "A" through "G" were used on the study. An aliquot of the test substance was analysed for purity in triplicate approximately at the beginning and end of each drum by gas chromatography. Purity results were compared to determine the test-substance stability.
The test material used in this study was greater than 99.9% pure. Samples taken from sample storage drums before and after the drums were opened for use in the test showed no evidence of change. Based on these results, the test substance was considered acceptable for use on the study and was considered stable over the duration of the study.

Test animals

Species:
rat
Strain:
other: Crl:CD®BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: (P) ca. 8 wks; (F1) ca. 4 wks
- Weight at study initiation: (P) Males: 266 g (mean value); Females: 190 g (mean value); (F1) Males: 68.5 g (mean value); Females: 63.5 g (mean value)
- Housing:
All male rats were housed individually during non-mating periods in stainless steel, wire-mesh cages.

a. Pretest and premating periods: All rats were housed individually in stainless steel, wire-mesh cages.
b. Cohabitation period: All rats were housed as breeding pairs in stainless steel, wire-mesh cages. Females without evidence of copulation by the end of the cohabitation period were housed individually in polycarbonate pans.
c. Gestation period
Days 0-14: Females were housed individually in stainless steel, wire-mesh cages.
Day 14-Delivery: Females were housed individually in polycarbonate pans.
d. Lactation period: Adult females were housed with their litters in polycarbonate pans.

The stainless steel, wire-mesh cages were suspended above cageboard. Polycarbonate pans contained bedding.
- Diet: Purina® Certified Rodent Checkers® was available ad libitum, except during exposures.
- Water: tap water was available ad libitum, except during exposures.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Atmosphere Generation: Atmospheres of cyclohexane were generated by metering the liquid test substance into a heated glass Instatherm flask with a Fluid Metering Inc. pump. Nitrogen, introduced into the flask, swept the cyclohexane vapour into the inhalation chamber air supply. The chamber concentration of cyclohexane was controlled by varying the amount of the metered liquid evaporated in the chamber air stream. Nitrogen and air were passed through the control chamber at approximately the same flow rates as those used in the exposure chambers (320 L/min). Chamber atmospheres were exhausted into the main plenum exhaust system and emitted into the atmosphere as allowed by permit.
The chamber distribution of cyclohexane vapour was determined prior to animal exposures in the high-concentration exposure chamber and while the study was underway with animals in the low- and high-concentration chambers. The results of these determinations indicated the distribution of cyclohexane vapour was sufficiently homogeneous (less than 1% difference in chamber concentration from position to position) for inhalation toxicology testing.
- Chamber Construction and Design: All exposure chambers (NYU style) were constructed of stainless steel and glass and had a nominal internal volume of 1.4 m³. The chamber volume was chosen so that the total body volume of the test animals did not exceed 5% of the chamber volume. A tangential feed at the chamber inlet promoted gas mixing and uniform chamber distribution of vapour.
- Environmental Monitoring: Chamber airflow was set to achieve at least 12 air changes per hour within the exposure chamber. Chamber temperature was targeted at 22 ± 2°C. Chamber relative humidity was targeted at 50 ± 10%. Chamber oxygen concentration was targeted to at least 19%. Airflow, temperature, and relative humidity were monitored continually and were recorded at 15-minute intervals during each exposure. Percent oxygen was recorded twice during each exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentration of cyclohexane was determined by gas chromatography generally at approximately 15-minute intervals during each exposure. Chamber atmosphere samples were directly injected into a Hewlett Packard model 5880 Gas Chromatograph equipped with a flame ionization detector for analysis of cyclohexane concentration. All samples were chromatographed isothermally at 70°C on an HP-20M Carbowax column.
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Until evidence of copulation was observed or until three weeks had elapsed.
- Proof of pregnancy: Intravaginal or extruded copulation plug referred to as Day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes (cohabitation period was 3 weeks, up to 3 pairing attempts were therefore conducted for unmated females)
- After successful mating each pregnant female was caged (how): individually in stainless steel, wire-mesh cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The atmospheric concentration of cyclohexane was determined by gas chromatography generally at approximately 15-minute intervals during each exposure. However, on test days 126 and 245, more than one hour elapsed between atmospheric analysis samples. Gas samples were drawn by vacuum pump from representative areas of the chamber where animals were exposed.
Chamber atmosphere samples were directly injected into a Hewlett Packard model 5880 Gas Chromatograph equipped with a flame ionization detector for analysis of cyclohexane concentration. All samples were chromatographed isothermally at 70°C on an HP-20M Carbowax column. The atmospheric concentration of cyclohexane was determined from a standard curve derived from gas standards. The gas standards were prepared prior to each exposure by injecting known volumes of liquid cyclohexane into gas bags that contained known volumes of air. The nominal atmospheric concentration was determined daily for each chamber by taking the total amount of cyclohexane delivered to the inhalation equipment divided by the total volume of air.
The mean analytically determined cyclohexane chamber concentrations were as targeted: 500 ± 0.63 (SEM), 2000 ± 2.8 (SEM) and 7000 ± 5.3 (SEM) ppm (2 significant figures). Day-to-day variability throughout the study was minimal. Nominal concentrations, calculated from pump set points and air flows, ranged up to 65% above the analytically determined concentrations but were considered to be within the limits of precision for the type of test chemical and inhalation systems used.
Duration of treatment / exposure:
(P) Males: 10 weeks before mating, 3 weeks during mating and ca. 3.5 weeks until sacrifice
(P) Females: 10 weeks before mating, 3 weeks during mating, 3 weeks during resulting pregnancies, 3 weeks through weaning of their F1 offspring (Days 5-24 of lactation only)
(F1) Males: 16-18 weeks at weaning, during growth into adulthood, mating and production of an F2 generation, until weaning of the F2 generation
(F1) Females: ca. 20.5 weeks at weaning, during growth into adulthood, mating and production of an F2 generation, until weaning of the F2 generation.
Frequency of treatment:
6 h/day, 5 days/week
Details on study schedule:
- F1 parental animals not mated until 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 25 days of age.
- Age at mating of the mated animals in the study: 15 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500, 2000 and 7000 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
ca. 1720, 6880 and 24080 mg/m³
Basis:
analytical conc.
based on MW = 84.16 g/mol
No. of animals per sex per dose:
30
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: The high-concentration level (7000 ppm) was selected on the bases of a pilot developmental toxicity study conducted at levels of 0, 3000, 6000, and 9000 ppm cyclohexane, and knowledge of the physical/chemical properties of cyclohexane. In the pilot developmental toxicity study, overall maternal body weight gain and food consumption were reduced during the exposure period at levels of 6000 ppm and above. Safety issues were integral to the selection of the high concentration. The high-concentration level was slightly less than 60% of the Lower Explosive Limit (LEL) of 1.2% (12000 ppm) for cyclohexane. The testing laboratory safety professionals judged this value to be the maximum value that permitted an adequate safety margin for personnel operating inhalation chambers of the size and type used in this study. The upper limit of 60% was selected based on safety standards set by the US National Fire Protection Association.
The low concentration (500 ppm) was selected because this value exceeds the threshold limit value (TLV) for human exposure (300 ppm) and was expected to cause no toxicological effects. The intermediate concentration level (2000) was selected to provide approximately equal spacing between the high and low concentrations on a log scale.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: mortality and abnormal behaviour and/or appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once weekly throughout the premating, gestation, and lactation periods for all P and F1 parental animals

CLINICAL OBSERVATIONS DURING EXPOSURE: Yes
- Procedure: During each exposure, rats were observed for mortality approximately every hour, and were assessed for their response to an alerting stimulus. The alerting response was determined prior to the initiation of each exposure, approximately two, four, and six hours after initiation of exposure, and during the time required to clear the chambers of test substance. Study technicians made a judgment whether the group of rats within a given exposure level displayed a normal, diminished, or hyperresponsive alerting behaviour in response to a standardized auditory stimulus.

BODY WEIGHT: Yes
- Time schedule for examinations:
Premating period: All P and F1 rats were weighed once a week.
Gestation period: P and F1 females were weighed on Gestation Days 0, 7, 14, and 21.
Lactation periods: P and F1 females were weighed on Lactation Days 0, 7. 14, 21 and
25.
Females, without evidence of copulation, or that copulated but did not deliver a litter, and males continued to be weighed weekly.

FOOD CONSUMPTION AND FOOD EFFICIENCY:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Body weight gain in g/food consumption in g for each study week calculated as
time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No
Sperm parameters (parental animals):
Parameters examined in all P and F1 male parental generations: testis weight
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.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after siring litters (Test Days 115-116 (P) and 111-129 (F1))
- Maternal animals: All surviving animals on Lactation Day 25 (P and F1). Non-pregnant P and F1 females were sacrificed on the same day as the corresponding pregnant females.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination: Testes (weighed), epididymides, prostate, seminal vesicles, coagulating gland, ovaries, uterus, vagina, cervix, pituitary and gross lesions.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 25 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
Collected gross lesions were prepared for microscopic examination.
Statistics:
The following statistical methods were applied for analysis of the indicated parameters:
- One-way Analysis of Variance; followed, when significant, with Dunnett's test: body weight, body weight gain, food consumption, food efficiency, gestation length and organ weight
- Cochran-Armitage test for trend: incidence of clinical observations, mating index, fertility index, gestation index and litter survival
- Jonckheere's test: implantation site numbers, implantation efficiency, mean number of pups per litter, sex ratio, percent born alive, viability index and lactation index
- Analysis of Covariance; followed, when significant, with linear contrast of the least square means: mean pup weights (covariates: litter size, sex ratio)
- Cochran-Armitage test for trend or, if not all groups are compared, Fisher's Exact test: incidence of microscopic lesions
For each parameter analyzed with a trend test, the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. For litter parameters, the proportion of affected pups per litter or the litter mean was used as the experimental unit for statistical evaluation. The level of significance selected was p < 0.05.
Where the data were tied and the standard large sample version of Jonckheere's test was not applicable, exact p values were calculated using permutation methodology.
Reproductive indices:
- Mating index (%) = (Number copulating/Number cohabited) x 100 (a)
- Fertility index (%) = (Number bearing litters/Number copulating) x 100 (b)
- Gestation index (%) = (Number of litters with at least one live pup/number of litters) x 100

(a) Number copulating: Evidence of copulation = copulatory plug, found dead pregnant, or delivery of a litter
(b) Number bearing litters: Including those found dead pregnant during gestation
Offspring viability indices:
- Implantation efficiency (%) = (Number of pups born/Number of implantations) x 100 (a)
- Pups born alive (%) = (Number of pups born alive/Number of pups born) x 100 (a)
- Viability index (%) = (Number of pups alive Day 4 preculling/Number of pups born alive) x 100 (a, b)
- Lactation Index (%) = (Number of pups alive at weaning on Day 25 post-partum/Number of pups alive Day 4 post-culling) x 100 (a, b)
- Litter survival (%): (Number of litters weaned/Number of viable litters delivered) x 100 (b)

(a) Determined for each litter. Mean and standard deviation for each dose level were calculated.
(b) Excluding litters sacrificed due to death of dam during lactation.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
≥ 2000 ppm: diminished or absent alerting responses during exposure (compound-related sedative effect)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
7000 ppm: statistically significant reduction in mean body weight (F1 males and P/F1 females), overall mean body weight gain (P/F1 females) and mean food consumption (P females during Gestation Days 0-7)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
7000 ppm: statistically significant reduction in mean body weight (F1 males and P/F1 females), overall mean body weight gain (P/F1 females) and mean food consumption (P females during Gestation Days 0-7)
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
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 (PARENTAL ANIMALS)
MORTALITY
There were no compound-related effects on mortality in rats of any generation.

CLINICAL SIGNS
Clinical observations during exposure
Compound-related effects on the alerting response determined during exposure were observed at 2000 and 7000 ppm. With a few exceptions (exposures 22-25 for the 2000 ppm group), animals exposed to 2000 or 7000 ppm exhibited a diminished response or no response during each exposure session, beginning exposures 16 and 15, respectively. The diminished or absent alerting responses were interpreted to be a compound-related sedative effect. Although the compound-related sedation was transient, it was considered to be toxicologically important.
There were a few spurious observations of a diminished response of animals during exposure to 500 ppm (exposures 102, 103, 162, 165, 166, 167 and 231). Those observations were considered not to be compound-related because of the lack of any consistency in this observation at this concentration. The diminished responses were not observed when the animals were at the smallest size (post-weaning) and, thus, receiving their highest dose. Nor did mean daily atmospheric analyses, indicating higher chamber concentration during the course of normal fluctuation, correlate with the days that the diminished responses were observed.
P males
There was a statistically significant increase in the incidence of the clinical signs "salivation, „stained perioral area" and “wet chin" observed in the P male rats exposed to 7000 ppm cyclohexane. The incidence of the clinical sign "stained perioral area" was also statistically significantly increased in the 2000 ppm P male rats.
The colour of each of the stains observed was brown and although the source of the stained fur was not obvious, it was presumed to be salivation. Those signs lasted only 10 to 15 minutes after the animals were removed from the chambers, but were not observed prior to exposure, nor during exposure. Similar clinical signs were observed in the 90-day rat study, 90-day neurotoxicity study with cyclohexane in rats, and in the developmental toxicity study in rats. In inhalation studies, rats are commonly observed to engage in grooming following cessation of test substance flow into the chambers. It is possible that the fur stains and wetness are related to this grooming activity. There were no adverse functional or morphological changes that could be correlated with the "stain chin", "wet chin" or "salivation”. Therefore, although the incidences of these signs in the 2000 and 7000 ppm groups were considered to be compound-related, they were considered not to be toxicologically important.
F1 males
There was a statistically significant increase in the incidence of the clinical signs "salivation", "stained chin" and "wet chin" observed in the F1 male rats exposed to 7000 ppm cyclohexane. The incidence of the clinical sign "stained chin" was also statistically significantly increased in the 2000ppm F1 male rats.
The nature of these signs were identical to those described for P males, and for the reasons discussed in the clinical observations section for P males, these signs observed in the 2000 and 7000 ppm groups were considered to be compound related, but were considered not to be toxicologically important.
P females
- Premating: there was a statistically significant increase in the incidence of the clinical signs “salivation”, “stained chin” and “wet chin” observed in the P female rats exposed to 7000 ppm cyclohexane.
- Gestation: the incidence of the clinical signs “stained chin” and “wet chin” was statistically significantly increased in the 7000 ppm P female rats.
- Lactation: the incidence of the clinical sign “stained chin” was statistically significantly increased for P females in the 7000 ppm group.
The nature of these signs were identical to those described for P males, and for the reasons discussed in the clinical observations section for P males, these signs observed in the 7000 ppm group were considered to be compound-related, but were considered not to be toxicologically important.
F1 females
- Premating: there was a statistically significant increase in the incidence of the clinical signs “salivation”, “stained chin” and “wet chin” observed in the F1 female rats exposed to 7000 ppm cyclohexane.
- Gestation: the incidence of the clinical signs “stained chin” and “wet perineum” was statistically significantly increased in the 7000 ppm F1 female rats.
- Lactation: the incidence of the clinical sign “stained chin” was statistically significantly increased for F1 females in the 7000 ppm group.
The nature of the signs “salivation”, “stained chin” and “wet chin” were identical to those described for P males, and for the reasons discussed in the clinical observations section for P males, the signs “salivation”, “stained chin” and “wet chin” observed in the 7000 ppm group were considered to be compound-related, but were considered not to be toxicologically important.
The statistically significantly increased incidence of “wet perineum“ in the 7000 ppm F1 females during gestation is considered not to be compound-related. The sign was observed sporadically across dose groups throughout the study. There was no other phase in this study in which the incidence of “wet perineum” resembled a dose-related pattern.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
P males
There were no compound-related statistically significant reductions in mean body weight or mean body weight gain for P male rats of any concentration in the study.
There was a statistically significant decrease (days 85-92 in the 500 ppm group) in mean body weight gain, which was considered not to be compound-related because of the absence of a dose response.
Both overall mean daily food consumption and overall mean food efficiency were comparable of that of controls.
There was a statistically significant decrease (days 1-8 in the 7000 ppm group) in mean food consumption, which was considered not to be biologically meaningful, because of the lack of any corresponding body weight effect. There was a statistically significant increase (days 29-36 in the 7000 ppm group) in mean food efficiency, which was considered not to be adverse.
F1 males
With the exception of test day 106, mean body weight was statistically significantly reduced throughout the study for F1 male rats in the 7000 ppm group. On test days 78 and 120, the mean body weight of the 7000 ppm group was approximately 94% of control
There was no statistically significant reduction in overall mean body weight gain for the premating period, although there was a statistically significant decrease in body weight gain for days 22-29 in the 7000 ppm group. Overall mean body weight gain for the entire period on test was mildly, but statistically significantly reduced for the 7000 ppm F1 males (approximately 94% of control).
The general lack of corresponding effects on body weight gain and food efficiency, suggests that the reduction in mean body weight observed in adult F1 male rats was probably due to pre-existing body weight deficits established as pups.
Mean daily food consumption and mean daily food efficiency did not differ significantly from controls for F1 male rats exposed to cyclohexane, during the premating period.
P females
- Premating: mean body weight was statistically significantly reduced for 7000 ppm P female rats by day 64 of the premating period. On test day 71, mean body weight for the 7000 ppm group was approximately 94% of that of the control group. Overall body weight gain for the premating period was statistically significantly reduced for P female rats in the 7000 ppm group (approximately 87% of control).
For P female rats, mean daily food consumption was not affected by exposure to cyclohexane. Overall mean daily food efficiency was statistically significantly lower than control rats for the premating phase (approximately 90% of control). There was a statistically significant increase in mean daily food consumption by P females in the 2000 ppm group for days 1-8 that was considered not to be compound-related due to the absence of a dose response.
- Gestation: mean body weight was statistically significantly reduced for P female rats in the 7000 ppm group throughout gestation (mean gestation body weights ranging from approximately 92 to 93% of control). However, there were no statistically significant differences in overall body weight gain for 7000 ppm P female rats during gestation, suggesting that the reduction in mean gestation body weight was probably due to pre-existing body weight deficits established during the premating period.
There was a statistically significant decrease in mean daily food consumption for gestation days 0-7 by the 7000 ppm pregnant females. Mean food efficiency, however, was comparable to control for treated female rats.
- Lactation: for lactation day 0, the mean body weight of 7000 ppm female rats was statistically significantly lower than that of the control. However, overall body weight gain for the 25-day lactation period was statistically significantly increased for the 7000 ppm group. Therefore, as with mean gestation body weight, the reduction in mean lactation body weight was probably due to pre-existing body weight deficits established during the premating period. Overall body weight gain for the 25-day lactation period was also statistically significantly increased for females exposed to 2000 ppm cyclohexane.
Mean daily food consumption by the 2000 ppm females was statistically significantly increased from lactation day 7 through the remainder of the lactation period. However, because of absence of a dose response, that increase was considered not to be compound-related. There was a statistically significant reduction in mean food efficiency for lactation days 21-25 for P females in the 7000 ppm group. Mean food efficiency was statistically significantly increased for lactation days 0-7 and 14-21 for 7000 ppm female rats, but the increase was considered not to be adverse.
F1 females
- Premating: mean body weight was reduced during the premating period for F1 female rats in the 7000 ppm group (statistically significantly reduced on days 1, 64 71 and 78). On day 1 of the premating period, the mean body weight of the 7000 ppm group was approximately 93% of control. At the end of the premating period on test day 78, the mean body weight of the 7000 ppm group was approximately 92% of control. Overall mean body weight gain for the premating period was statistically significantly lower for 7000 ppm F1 females (approximately 92% of control).
- Gestation: mean body weight for 7000 ppm F1 female rats was statistically significantly reduced throughout gestation. However, with the exception of mean body weight gain of 7000 ppm females for gestation days 0-7, there were no statistically significant differences in mean body weight gain or overall gestation body weight gain in F1 females exposed to cyclohexane compared to control. As in the P generation, the reduction in mean gestation body weight was probably a continuation of pre-existing body weight deficits established during the premating period.
- Lactation: the mean body weight of lactating 7000 ppm female rats was statistically significantly lower than that of the control throughout the 25-day lactation period. However, overall body weight gain for the 25-day lactation period was greater for the 7000 ppm group than for controls. Therefore, as in the P generation, the reduction in mean lactation body weight was most likely due to pre-existing body weight deficits established during the premating period.
Overall mean daily food consumption of treated groups was comparable to controls during the F1 premating, gestation and lactation phases. Overall mean food efficiency for the F1 premating phase was statistically significantly reduced for 7000 ppm F1 females. There was also a statistically significant reduction in mean food efficiency for gestation days 0-7 for F1 females in the 7000 ppm group. Mean daily food efficiency of treated groups was comparable to controls during the F1 lactation phase.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no biologically or statistically significant dose-related differences in mating, fertility or gestation indices, implantation efficiency or gestation length in either the P or F1 generation.
In general, fertility index for the F1 generation was somewhat low. However, fertility index values for each of the treated groups were higher than that of the concurrent control group and the value for the concurrent control group (73.3) was above the mean control data from previous studies conducted at the test facility over the past few years (mean fertility index ± standard deviation: 72.4 ± 9.8; range: 58.3-86.2).

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no compound-related effects on testes weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no compound-related gross observations in rats of any generation. Observations occurred in low incidences and were randomly distributed across control and treatment groups.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no compound-related microscopic findings in rats of any generation. There was a slight increase in the incidence of prostatic inflammation in the 7000 ppm P and F1 males when compared to controls. The severity of this lesion was only greater than minimal in four P and F1 control and four high-dose males. Prostatic inflammation is a common microscopic change observed in male rats and is predominantly found in the dorsolateral prostate. This microscopic finding was considered an incidental finding due to its lack of severity and reported common occurrence in this species. Other lesions occurred in low incidences without a relevant dose-response relationship and were considered incidental occurrences of spontaneous lesions in rats of this strain and age and unrelated to administration of cyclohexane.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
2 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight (decreased pup weights in both the F1 and F2 generations observed at the highest concentration)
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
6 880 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on MW = 84.16 g/mol
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
NOAEC
Remarks:
systemic toxicity (parental)
Effect level:
500 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs (sedative effects during exposure at the mid- and high concentrations)
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEC
Remarks:
systemic toxicity (parental)
Effect level:
1 720 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on MW = 84.16 g/mol
Remarks on result:
other: Generation: P and F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
7000 ppm: statistically significant reduciton in in pup body weight from postpartum day 7 throughout the remainder of the 25-day lactation period for F1 litters and F2 litters
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

LITTER SIZE AND PUP SURVIVAL
With the exception of the mean percent born alive of F1 litters, there were no statistically significant dose-related trends in mean number of implantation sites, mean number of pups/litter or any survival indices (sex ratio, percent born alive, 0-4 day viability, lactation index and litter survival) for both F1 and F2 litters throughout lactation. For F1 litters of 7000 ppm females, mean percent born alive was statistically significantly reduced, however, that reduction was considered not to be adverse since the value (98.1%) fell above the mean of the historical control data of the test facility (mean F1 % pups born alive ± standard deviation: 97.5 ± 2.0; range: 92.5-100).

PUP WEIGHTS
The mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for F1 litters and F2 litters of the 7000 ppm group.

CLINICAL OBSERVATIONS IN PUPS
There were no statistically significant dose-related differences in the incidences of any clinical signs among F1 or F2 litters.

GROSS PATHOLOGY
There were no compound-related gross observations in rats of any generation. Observations occurred in low incidences and were randomly distributed across control and treatment groups.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A Two-Generation Reproduction Toxicity study was conducted with cyclohexane according to OECD Guideline 416 (adopted 1983) and under GLP conditions. The study involved the production of one set of litters in each generation. Throughout the study, rats (30 per sex and concentration) were exposed whole-body to clean air or atmospheric concentrations of 500, 2000 or 7000 ppm cyclohexane (corresponding to 1720, 6880 and 24080 mg/m³, respectively), 6 h/day, 5 days/week. Following at least 10 weeks of exposure, the animals were bred within their respective treatment groups, and allowed to deliver and rear their offspring until weaning (postpartum Day 25). At weaning, 30 F1 rats per sex and group were randomly selected to produce the next generation. At least 11 weeks after weaning, the F1 rats were bred within their respective treatment groups to produce F2 litters. Barring F1 weanlings selected for propagation of the next generation, twenty F1and F2 weanlings per sex and concentration were randomly designated for gross postmortem examination. The remaining unselected weanlings were sacrificed without pathological examination.
At the 7000 ppm level, adverse effects considered to be related to cyclohexane treatment included statistically significant reductions in mean body weight and overall mean body weight gain for P and F1 females; a statistically significant reduction in mean body weight for F1 males; statistically significant reductions in overall mean food efficiency for P anf F1 females during premating and in mean food efficiency for Lactation Days 21-25 for F1 females; statistically significant reductions in mean food consumption by P females for Gestation Days 0-7; Statistically significantly reduced mean pup weight from Lactation Day 7 throughout the remainder of the 25-day lactation period for F1 and F2 litters; generally diminished response or no response of the animals to a sound stimulus while in the chambers during exposure.
At the 2000 ppm level, adverse effects considered to be related to cyclohexane treatment included generally diminished response or no response of the animals to a sound stimulus while in the chambers during exposure.
No adverse compound-related effects were observed at 500 ppm. Therefore, the systemic NOAEC was 500 ppm (1720 mg/m³) and the reproductive NOAEC was 2000 ppm (6880 mg/m³) in male and female rats. The reproductive NOAEC was based solely on the decreased pup weights in both the F1 and F2 generations observed at 7000 ppm (24080 mg/m³).