N-hexane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity data is available for n-hexane and commercial hexane and presented in the dossier.

 

Commercial hexane

Two-Generation Reproduction Toxicity Study (OECD TG 416)

Inhalation NOAEC (Reproductive toxicity) = 31680 mg/m³ air

n-Hexane

Inhalation LOAEC (Testicular effects): 17600 mg/m³

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-09-18 to 1990-06-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restriction because it followed a protocol comparable to OECD Guideline 416.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: (P) 28 days; (F1) 29-31 days
- Weight at study initiation: (P) Males: 75-100 g; Females: 65-80 g
- Housing: individually except during mating and lactation in stainless steel wire mesh cages, females were housed in plastic cages from gestational day (GD 20) through weaning; animals were identified by ear notches or toe clips
- Diet (e.g. ad libitum): Certified Ground Rodent Diet RMH 3200, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-73 degree F
- Humidity (%): 40-63
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: From: Sept. 18, 1989 To: June 16, 1990

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 900 l glass and stainless steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: 200 l/min
- Method of conditioning air: Test substance was metered from a piston pump into a heated glass evaporator with a temperature of 36-61 degree C. Conditioned air was passed through the evaporator, where it carried the vapor into the exposure chamber.
- Temperature, humidity: monitored every 30 minutes
- Air flow rate: 200 l/min
- Air change rate: 20 min
- Treatment of exhaust air: filtration


TEST ATMOSPHERE
- Brief description of analytical method used: GC with flame ionization detection
- Samples taken from breathing zone: yes, six times per exposure

Details on mating procedure:

- M/F ratio per cage: 1/1 - If mating failed, females were switched to the male of an unmated pair in the same dose group after 7 days. If mating failed again, they were switched after another 7 days.
- Length of cohabitation: 3 weeks, including during exposure
- Proof of pregnancy: vaginal plug, day 0

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken six times per exposure period and analyzed with GC-FID. Distribution of test substance was evaluated by sampling nine different areas of the exposure chamber.

Duration of treatment / exposure:

10 weeks pre-breeding, 3 weeks during breeding
Females continued to be exposed through GD 19. Exposure was resumed on postnatal day 5, and continued through weaning.
The F1 generation was treated similarly, but pre-breeding exposure was 8 weeks.

Frequency of treatment:

6 hrs/day, 5 days/week

Details on study schedule:

- F1 parental animals not mated until 9 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 13-16 weeks

Remarks:

Doses / Concentrations:
0, 900, 3000, 9000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
892, 2995, 9019 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

25 per sex per dose

Control animals:

yes, sham-exposed

Details on study design:

none provided

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, toxicity, littering, mating

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption: Yes, food consumption of pregnant females was measured in 3-4 day intervals, and through postnatal day 28.

Litter observations:

STANDARDISATION OF LITTERS
Parents of the F2 generation were selected on day 28 postpartum, at least one pup per litter was selected, with a second pup selected only if all litters were already represented. The F2 generation was standardized on day 4 postpartum.


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 abnormalities


GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after parturition of the first litter
- Maternal animals: All surviving animals day after weaning.


GROSS NECROPSY
- Gross necropsy consisted of external surfaces, orifices, cranial cavity, carcass, brain, spinal cord, thoracic cavity, abdominal cavity, pelvic cavity, cervical tissues and organs

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues from 25 male and females from the high dose and control groups were examined including testes of males failing to mate.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 days of age.
- These animals were subjected to postmortem examinations as follows: stillborn and pups dying during lactation, culled pups


GROSS NECROPSY
- Gross necropsy consisted of external examinations.

Statistics:

Quantitative continuous variables were compared by use of Levene's test for equal variance, analysis of variance, and t-tests. Significance for t-tests were corrected by the Bonferroni method. Nonparametric data was evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney test. Indices were compared using Fisher's exact test. 0.05 was used as the criteria for statistical significance.

Reproductive indices:

mating index, fertility index, gestational index, live birth index,

Offspring viability indices:

4-day survival index, 7-day survival index, 14-day survival index, 21-day survival index, lactation index

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

F0 GENERATION

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related effects observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no treatment related effects to food consumption. Males in the 9000 ppm group had reduced body weight during week 13. Body weight gains in this group were reduced during weeks 7, 11-12, and 12-13. Males in the 3000 ppm group had reduced body weight gain in weeks 4-5, and reduced weight in weeks 9-10. Females weight gains were reduced in the 9000 ppm group in weeks 5-6.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Lactational food consumption was significantly reduced during days 7-11, and days 19-21 in the 9000 ppm group. No other reproductive parameters differed significantly from controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment related abnormalities were seen.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Hyaline droplet nephropathy and tubular basophilia were seen in the 9000 ppm males.

F1 GENERATION
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related effects were observed. One female in the 900 ppm group died on day 83 due to prolonged delivery.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights of 9000 ppm males were significantly reduced throughout the exposure period. Weight gain was reduced in this group during the weeks 9-10, and 10-11. Females in the 9000 ppm group had reduced body weight during the first 3 weeks of pre-breeding exposure.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In the 9000 ppm group, food consumption was reduced on gestational days 0-4, and 4-7, and gestational intervals 0-7, and 7-14. This group also had reduced food consumption during lactational days 21-24, 26-27, 21, and 28. In the 3000 ppm group, food consumption was reduced during lactational days 22-23, and in the 900 ppm group during days lactational days 21-22.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment related abnormalities were seen.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Hyaline droplet nephropathy and tubular basophilia were seen in the 9000 ppm males.

Key result

Dose descriptor:

NOAEC

Effect level:

3 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: reduced body weight

Remarks on result:

other: Generation: F1, F2

Dose descriptor:

LOAEC

Effect level:

9 000 ppm

Sex:

male/female

Basis for effect level:

other: reduced body weight

Remarks on result:

other: Generation: F1, F2

Key result

Dose descriptor:

NOAEC

Effect level:

9 000 ppm

Sex:

male/female

Remarks on result:

other: Generation: reproductive toxicity

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

F1 GENERATION
VIABILITY (OFFSPRING)
The number of dead pups was increased in the 900 ppm exposure group, however, as this was not seen at higher doses, it was not considered treatment related.

BODY WEIGHT (OFFSPRING)
The body weight of pups in the 9000 ppm group were reduced beginning on lactational day 14. Body weight gains in this group were reduced during lactational days 14-21 for females, and lactational days 7-14 for all pups.


GROSS PATHOLOGY (OFFSPRING)
No treatment related effects were noted.

F2 GENERATION
VIABILITY (OFFSPRING)
Viability was unaffected by exposure.

BODY WEIGHT (OFFSPRING)
The body weight of pups in the 9000 ppm group were from lactational day 7-28. Body weight gains in this group were reduced during lactational days 14-21 for females, and lactational days 7-14 for all pups. There were significantly reduced body weight gains in pups in the 9000 ppm group during lactational days 4-7, and 7-14, and slightly reduced weight gains on lactational days 14-21.

GROSS PATHOLOGY (OFFSPRING)
No treatment related effects were noted.

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

3 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: reduced body weight

Remarks on result:

other: Generation: F1, F2

Key result

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

9 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: reduced body weight

Remarks on result:

other: Generation: F1, F2

Reproductive effects observed:

not specified

Significant Results of Reproductive Toxicity Study on Rats

Concentration (ppm)	0	900	3000	9000
Body weight of F0 adult males - week 13 (g)	463.7 (48.93)	455.2 (34.22)	455.2 (40.25)	436.1 (24.83)
Body weight gain of F0 adult males - week 4-5 (g)	32.6  (8.98)	28.9 (8.56)	24.2 (7.89)	28.9 (3.78)
Body weight gain of F0 adult males - week 6 -7 (g)	25.4 (6.17)	25.4 (6.28)	23.7 (4.94)	21.2 (4.31)
Body weight gain of F0 adult males - week 9-10 (g)	24.2 (6.00)	21.6 (6.07)	18.6 (6.82)	19.9 (6.17)
Body weight gain of F0 adult males - week 11-12 (g)	11.9 (5.40)	10.7 (6.51)	12.7 (4.83)	3.3 (5.70)
Body weight gain of F0 adult males - week 12-13 (g)	11.8 (6.26)	7.4 (6.34)	8.7 (7.28)	6.4 (6.09)
Body weight gain of F0 adult females - week 0-1 (g)	0.3 (3.08)	3.4 (3.25)	1.9 (2.74)	0.8 (3.67)
Body weight gain of F0 adult females - week 5-6 (g)	11.8 (4.01)	11.0 (4.40)	12.3 (3.57)	9.0 (3.20)
Lactational food consumption F0 - day 7-11 (g/animal/day)	44.63 (3.859)	42.93 ()	43.54 (3.796)	41.45 (3.244)
Lactational food consumption F0 - day 19-21 (g/animal/day)	64.41 (5.833)	64.87 (5.439)	62.32 (6.595)	59.81 (8.212)
No. dead F1 pups - lactational day 4	5	26	12	7
F1 pup body weight - lactational day 21 (g)	41.93 (3.950)	42.50 (4.125)	39.97 (3.292)	38.92 (3.996)
F1 female pup body weight - lactational day 21 (g)	41.48 (4.151)	41.75 (4.168)	39.52 (3.430)	38.10 (4.063)
Body weight changes in F1 pups - lactational day 7-14 (g)	11.91 (1.617)	12.11 (1.328)	11.48 (1.381)	10.56 (1.780)
Body weight changes in F1 male  pups - lactational day 7-14 (g)	12.00 (1.628)	12.24 (1.306)	11.41 (1.708)	10.71 (1.847)
Body weight changes in F1 female pups - lactational day 7-14 (g)	11.81 (1.677)	12.00 (1.420)	11.51 (1.536)	10.35 (1.789)
Body weight changes in F1 female pups - lactational day 14-21 (g)	15.86 (1.933)	15.47 (2.162)	14.39 (1.744)	14.24 (2.343)
Food consumption in F1 females - week 0-1 (g/animal/day)	20.9 (1.87)	20.9 (2.00)	20.7 (2.68)	19.0 (1.62)
Food consumption in F1 females - week 1-2 (g/animal/day)	21.5 (1.45)	21.2 (2.29)	21.2 (2.80)	19.1 (1.90)
Food consumption in F1 females - week 3-4 (g/animal/day)	22.0 (2.40)	21.8 (2.74)	21.5 (2.98)	19.6 (1.99)
Food consumption in F1 females - week 5-6 (g/animal/day)	20.8 (2.02)	21.2 (2.60)	20.6 (2.87)	19.1 (2.00)
Food consumption in F1 females - week 7-8 (g/animal/day)	20.3 (1.84)	20.3 (2.24)	20.0 (2.37)	18.4 (1.99)
F1 Gestational food consumption - day 0-4 (g/animal/day)	22.87 (3.172)	21.93 (2.407)	21.93 (3.237)	19.67 (1.703)
F1 Gestational food consumption - day 4-7 (g/animal/day)	24.31 (3.047)	23.63 (3.228)	23.42 (3.077)	21.81 (2.072)
F1 Gestational food consumption - day 0-7 (g/animal/day)	23.48 (2.972)	22.44 (2.503)	22.57 (2.905)	20.56 (1.760)
F1 Gestational food consumption - day 7-14 (g/animal/day)	26.28 (3.268)	25.25 (3.108)	24.52 (3.055)	23.70 (2.565)
F1 lactational food consumption - day 21-22 (g/animal/day)	87.77 (15.326)	79.55 (8.381)	80.31 (8.272)	74.01 (9.711)
F1 lactational food consumption - day 22-23 (g/animal/day)	91.26 (10.218)	87.42 (9.649)	83.36 (8.764)	81.23 (10.532)
F1 lactational food consumption - day 23-24 (g/animal/day)	97.23 (11.339)	94.59 (9.185)	90.30 (6.703)	85.17 (13.188)
F1 lactational food consumption - day 26-27 (g/animal/day)	115.86 (11.445)	114.19 (16.261)	109.85 (11.689)	105.38 (15.023)
F1 lactational food consumption - day 21-28 (g/animal/day)	102.87 (7.787)	100.49 (8.471)	97.47 (6.852)	94.04 (10.541)

Conclusions:

The NOAEC for both male and female rats (adults and offspring) was 3000 ppm (10560 mg/m3). The LOAEC for these groups was 9000 ppm based on reduced body weight. There were no adverse effects to reproduction, therefore the NOAEC for reproduction is 9000 ppm (31680 mg/m3).

Executive summary:

The purpose of this study was to determine the effect of commerical hexane on reproduction in rats. Groups of 25 male and 25 female rats were exposed to nominal concentrations of 0, 900, 3000, or 9000 ppm of test substance for 10 weeks pre-breeding, 3 weeks during breeding, and postnatal days 4 -28. After weaning, pups were selected to be parents for the F2 generations, and treated similarly to their parents, except their pre-breeding exposure was 8 weeks. During exposure, animals were monitored for mortality, clinical signs, food consumption, and body weight. Offspring were examined for body weight, survival, and viability. Both parents and offspring were sacrificed and examined for gross abnormalities, and in the case of adults histopathology. Reproductive parameters were similar in exposure groups and control groups. There was reduced body weight in the F1 and F2 generation in both sexes in the 9000 ppm exposure group in both adults and offspring. The NOAEC is therefore 3000 ppm (10560 mg/m3), and the LOAEC is 9000 ppm (31680 mg/m3). Since there were no adverse effects in offspring without adverse maternal effects, the NOAEC for reproduction is 9000 ppm (31680 mg/m3).

Reference 2

Endpoint:

reproductive toxicity, other

Remarks:

other: acute inhalation toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable with restrictions because there was no GLP statement provided, and limited data on methods were reported, but the study seemed to be well-conducted.

Qualifier:

no guideline required

Guideline:

other: OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Italy
- Weight at study initiation: 180-220 g
- Housing: steel wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: airtight Lucite boxes
- Method of holding animals in test chamber: cages
- Source and rate of air: 3 l/min
- Method of conditioning air: Air from a compressed air bottle and regulated by a flowmeter conveyed n-hexane vapor from a bubbler immersed in a 23.5 degree C water bath. This air mixed with air from a pump in a glass chamber before entering the control chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: A total hydrocarbon analyzer was connected to the exhaust from exposure group chambers.

Details on mating procedure:

Not applicable

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

(1) a single 24-hour exposure, (2) repeated 16-hour/day exposures for up to 8 days, or (3) repeated 16-hour/day exposures, 6 hours/day for up to 6 weeks.

Remarks:

Doses / Concentrations:
5000 ppm
Basis:

No. of animals per sex per dose:

12-39 per group

Control animals:

other: pair fed

Details on study design:

- Dose selection rationale: not provided

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

Not applicable

Sperm parameters (parental animals):

Parameters examined in males: epididymyal tubules, testicular lesions

Litter observations:

Not applicable

Postmortem examinations (parental animals):

Not provided

Postmortem examinations (offspring):

not applicable

Statistics:

Body weight data was analyzed using the Student t-test.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): The animals exposed for up to 6 weeks displayed a reduction in food consumption and body weight gain; these effects were accompanied by signs of incipient neuropathy.

Dose descriptor:

other: LC50

Effect level:

> 5 000 ppm

Based on:

test mat.

Sex:

male

Remarks on result:

other: Generation: not applicable

Key result

Dose descriptor:

LOAEC

Remarks:

24 hour

Effect level:

> 5 000 ppm

Sex:

male

Remarks on result:

other: Generation: not applicable

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Not applicable

Remarks on result:

other: Generation not applicable

Reproductive effects observed:

not specified

Conclusions:

There was no mortality of animals due to a single 24-hr exposure to 5000 ppm test substance. Rats receiving a single 24-hour exposure to n-hexane also showed a measure of recovery after 2–4 weeks following the termination of exposure. By contrast, rats exposed repeatedly to 5000 ppm n-hexane over a 6-week period showed complete atrophy of the seminiferous tubules. The animals exposed for up to 6 weeks displayed a reduction in food consumption and body weight gain; these effects were accompanied by signs of incipient neuropathy. There was also a wide range of testicular lesions that did not completely resolve during the recovery period even though body weights and clinical symptoms improved. The LC50 is > 5000 ppm for male rats.

Executive summary:

In an acute inhalation toxicity study researchers exposed male Sprague-Dawley rats (12–39/group) to 5000 ppm n-hexane (99% pure) in either (1) a single 24-hour exposure, (2) repeated 16-hour/day exposures for up to 8 days, or (3) repeated 16-hour/day exposures, 6 hours/day for up to 6 weeks. Two control groups were employed, one of which was pair-fed. Treated animals were allowed to recover for different lengths of time after the end of treatment (from 2 days to 29 weeks, depending on the original exposure duration).

Rats exposed to 5000 ppm n-hexane displayed some evidence of neuropathy such as paralysis, and extreme cases were sacrificed moribund and necropsied rather than being allowed to die and undergo partial autolysis. Focal degeneration of spermatocytes and exfoliation of elongated spermatids was observed in rats treated with n-hexane. Early meiotic prophase spermatocytes (leptotene and zygotene) and transitional spermatocytes as well as those undergoing meiotic metaphase appeared to be more susceptible to the action of n-hexane than pachytene spermatocytes.

After the 24-hr treatment was suspended, damage to the seminiferous epithelium increased for the first 7 days, while the epididymis also exhibited focal infiltration by inflammatory cells; recovery was completed from Days 14 to 30. Animals exposed for 16 hr/day, 6 days/week at the same concentration of 5000 ppm for up to 6 weeks induced progressive increases in testicular and epididymal lesions, which, after 5 weeks (when most animals began to show clinical symptoms of polyneuropathy), reached aplasia of the germinal epithelium involving also the spermatogonia. A fter interruption of the treatment, the testicular lesions became increasingly severe, up to complete atrophy of the seminiferous tubules, which suggests irreversible sterility.

Reference 3

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Data waiving:

other justification

Justification for data waiving:

the study does not need to be conducted because a two- (or multi-) generation reproductive toxicity study is available

other:

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEC

17 600 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

96

Species:

rat

Quality of whole database:

2 key studies available for assessment.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity data is available for n-hexane and commercial hexane and presented in the dossier.

 

Inhalation

 

n-hexane

In a key study (De Martino et al. 1987), rats exposed via inhalation for 16 hr/day, 6 days/week at 5000 ppm (17600 mg/m3) n-hexane for 6 weeks induced progressive increases in testicular and epididymal lesions, which, after 5 weeks (when most animals began to show clinical symptoms of polyneuropathy), reached aplasia of the germinal epithelium involving also the spermatogonia. After interruption of the treatment, the testicular lesions became increasingly severe, up to complete atrophy of the seminiferous tubules, which suggests irreversible sterility.

 

Commercial hexane

In a key reproduction toxicity study the effect of inhalation of commercial hexane (52% n-hexane) on reproduction in rats was determined (Daughtrey, 1994). Groups of 25 male and 25 female rats were exposed to nominal concentrations of 0, 900, 3000, or 9000 ppm of commercial hexane for 10 weeks pre-breeding, Reproductive parameters were similar in exposure groups and control groups. There was reduced body weight in the F1 and F2 generation in both sexes in the 9000 ppm exposure group in both adults and offspring. The NOAEC is therefore 3000 ppm (10560 mg/m3), and the LOAEC is 9000 ppm (31680 mg/m3). Since there were no adverse effects in offspring without adverse maternal effects, the NOAEC for reproduction is 9000 ppm (31680 mg/m3).

Effects on developmental toxicity

Description of key information

Developmental toxicity data is available for n-hexane.

 

Developmental Toxicity Study (rat) - Inhalation Administration

Developmental NOAEC = 200 ppm (704 mg/m³).

 

Developmental Toxicity Study (mouse) - Inhalation Administration

Developmental LOAEC = 200 ppm (704 mg/m³).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable with restrictions because there was no GLP statement provided, and limited data on methods were reported, but the study seemed to be well-conducted.

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study examined the effect of inhalation of n-hexane vapors to fetal development. Groups of 35 pregnant and 10 virgin female mice were exposed to concentrations of 0, 200, 1000, or 5000 ppm of test substance vapors for 20 hrs/day, daily, during gestational days 6 -17. During exposure, females were weighed regularly. At the end of the exposure period, the females were sacrificed, and the uterine contents were examined for parameters including number of live fetuses, number of implantations, and number of resorptions. Fetuses were also examined for malformations.

GLP compliance:

no

Limit test:

yes

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Raleigh NC
- Weight at study initiation: 28.7-31.5 g
- Housing: cages
- Diet (e.g. ad libitum): NIH-07 diet, ad libitum
- Water (e.g. ad libitum): ad libitum



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 73 +/- 3 degrees F
- Humidity (%): 50 +/- 15 %
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Battelle-designed chambers, 2.3 m3 stainless steel chambers
- Method of holding animals in test chamber: cages
- Method of conditioning air: Hexane in a 19 l stainless steel reservoir is pumped into delivery tubes to vaporizers at the fresh air inlet to the exposure chambers. The vaporizers are heated to aid in evaporation. Micrometering pumps are used to adjust the concentration.
- System of generating particulates/aerosols:
- Temperature, humidity in air chamber: 73 +/- 3 degree F, 50 +/- 15%
- Air change rate: approx. 15 changes per hour
- Treatment of exhaust air: building exhaust


TEST ATMOSPHERE
- Brief description of analytical method used: Atmosphere was tested using a on-line GC with automatic 8-port sampling valve.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Atmosphere was tested using a on-line GC with automatic 8-port sampling valve.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male to 1-2 females
- Length of cohabitation: up to five nights
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

20 hrs/day

Frequency of treatment:

daily

Duration of test:

gestation days 6-17

Remarks:

Doses / Concentrations:
0, 200, 1000, 5000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

35 pregnant females, 10 virgins per dose

Control animals:

yes, sham-exposed

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: plug positive females - days 0, 6, 9, 12, and 18
virgin females - 12 day prior to exosure, days 1, 4 and 7, before sacrifice


POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 20
- Organs examined: nongravid uteri

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

SAS statistical software, Duncan's multiple range test
Mean body weigths: SAS General Linear Models Procedure with analysis of variance
Dose response: orthogonal trend test

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Mean weight gain for pregnant females in the 5000 ppm group was significantly reduced compared to controls. There was also a significant reduction in gravid uterine weight in the 200 and 5000 ppm groups. A significant reduction in uterine weight to extra-gestational weight gain was seen in the 5000 ppm group as well. Number of implantations per litter was also reduced in the 5000 ppm group, and also the number of live implants per litter. An increase in intrauterine death was significantly greater only in the 200 ppm group. The incidence of late resorptions was significantly increased in the 5000 ppm group.

Key result

Dose descriptor:

NOAEC

Effect level:

1 000 ppm

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEC

Effect level:

5 000 ppm

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

LOAEC

Effect level:

200 ppm

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal weights of females in the 5000 ppm group were significantly reduced. The only significant increase in malformations as compared to controls, was an exencephalic fetus in the 5000 ppm group, however, as this was only seen in one fetus, and this malformation was also present in the control group, it is not considered treatment related. Rather it is an anomalous result due to the low background incidence of this malformation.

Key result

Dose descriptor:

LOAEC

Effect level:

200 ppm

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Significant results of mouse developmental toxicity study

Concentration	0 ppm	200 ppm	1000 ppm	5000 ppm
Mean body weight – gestational day 18 (g)	57.0 ± 5.0	54.8 ± 4.0	55.7 ± 4.9	53.7 ± 4.6
Uterine mean weight (g)	21.4 ± 3.8	19.2 ± 3.4	20.2 ± 3.5	17.8 ± 3.9
Live fetuses/litter	12.0 ± 2.6	10.6 ± 2.2	11.4 ± 2.4	10.2 ± 2.7
Resorptions/litter	0.6 ± 0.7	1.6 ± 1.7	0.8 ± 0.9	1.1 ± 1.2
Late resorptions/litter	0.0 ± 0.2	0.4 ± 0.9	0.4 ± 0.6	0.5 ± 0.8
% live fetuses/litter	95.5 ± 5.6	87.2 ± 12.2	93.5 ± 7.2	89.7 ± 12.4
% resorptions/litter	4.5 ± 5.6	12.8 ± 12.2	6.5 ± 7.2	10.3 ± 12.4
% late resorptions/litter	0.3 ± 1.3	3.4 ± 6.6	2.9 ± 4.6	4.5 ± 6.5
Female fetal weight (g)	1.39 ± 0.10	1.37 ± 0.10	1.35 ± 0.10	1.31 ± 0.11
% exencephaly/litter affected	0 ± 0	0 ± 0	0 ± 0	0.5 ± 2.5

Conclusions:

The maternal NOAEC was 1000 ppm, and the maternal LOAEC was 5000 ppm based on reduced body weight. The developmental LOAEC was 200 ppm (704 mg/m3) based on increased resorptions and reduced uterine weight. There was no NOAEC for developmental toxicity.

Executive summary:

This study examined the effect of inhalation of n-hexane vapors to fetal development. Groups of 35 pregnant and 10 virgin female mice were exposed to concentrations of 0, 200, 1000, or 5000 ppm of test substance vapors for 20 hrs/day, daily, during gestational days 6 -17. During exposure, females were weighed regularly. At the end of the exposure period, the females were sacrificed, and the uterine contents were examined for parameters including number of live fetuses, number of implantations, and number of resorptions. Fetuses were also examined for malformations. Body weight was significantly reduced in 5000 ppm exposure females. There was significant reduction in gravid uterine weight in the 200 ppm group, there was also an increase in intrauterine death in this group. Fetal weights were also reduced in the 5000 ppm group. No biologically significant increase in malformations was found. The NOAEC for maternal toxicity is 1000 ppm, and the LOAEC is 5000 ppm based on reduced body weight. There was no NOAEC found for developmental toxicity, and the LOAEC is 200 ppm (704 mg/m³) based on reduced fetal weight gain.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable with restrictions because there was no GLP statement provided, and limited data on methods were reported, but the study seemed to be well-conducted.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 30 pregnant and 10 virgin female rats were exposed to concentrations of 0, 200, 1000, or 5000 ppm of test substance vapors for 20 hrs/day, daily, during gestational days 6 -20. During exposure, females were weighed regularly. At the end of the exposure period, the females were sacrificed, and the uterine contents were examined for parameters including number of live fetuses, number of implantations, and number of resorptions. Fetuses were also examined for malformations.

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Raleigh, NC
- Weight at study initiation: 232.4-323.8 g
- Housing: 5 same sex animals per cage
- Diet (e.g. ad libitum): NIH-07 diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 20 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 73 +/- 3 degree F
- Humidity (%): 50 +/- 15%
- Air changes (per hr): 14.3-15.3 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Battelle-designed chambers, 2.3 m3 stainless steel chambers
- Method of holding animals in test chamber: cages
- Method of conditioning air: Hexane in a 19 l stainless steel reservoir is pumped into delivery tubes to vaporizers at the fresh air inlet to the exposure chambers. The vaporizers are heated to aid in evaporation. Micrometering pumps are used to adjust the concentration.
- System of generating particulates/aerosols:
- Temperature, humidity in air chamber: 73 +/- 3 degree F, 50 +/- 15%
- Air change rate: approx. 15 changes per hour
- Treatment of exhaust air: building exhaust


TEST ATMOSPHERE
- Brief description of analytical method used: Atmosphere was tested using a on-line GC with automatic 8-port sampling valve.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Atmosphere was tested using a on-line GC with automatic 8-port sampling valve.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 2 females/1 male
- Length of cohabitation: up to 4 nights
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

20 hrs

Frequency of treatment:

daily

Duration of test:

Gestation days 6-20

Remarks:

Doses / Concentrations:
0, 200, 1000, 5000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

30 pregnant dams, 10 virgin females

Control animals:

yes, sham-exposed

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: sperm-positive females - days 0, 6, 13, and 20
virgin females - 14 day prior to exposure, days 1 and 8, before sacrifice


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: nongravid uteri

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

SAS statistical software, Duncan's multiple range test
Mean body weigths: SAS General Linear Models Procedure with analysis of variance
Dose response: orthogonal trend test

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
2 pregnant dams in the 2000 ppm group, one non-pregnant dam in the 200 ppm group, and one virgin in the 5000 ppm group showed ulceration at the time of sacrifice. Virgin females in the 5000 ppm group had significantly reduced body weight for 14 consecutive days. A significant reduction in body weight was seen in 5000 ppm pregnant females as well. Body weight gain was reduced significantly for pregnant females in the 1000 ppm group on day 20, and 5000 ppm group at days 13 and 20. A significant reduction in gravid uterine weight was seen in females in the 5000 ppm group. Extra-gestational weight gain was reduced for females in the 5000 ppm group significantly.

Key result

Dose descriptor:

NOAEC

Effect level:

200 ppm

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEC

Effect level:

1 000 ppm

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEC

Effect level:

200 ppm

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEC

Effect level:

1 000 ppm

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There was a significant reduction in fetal body weights in the 1000 and 5000 ppm groups. Male fetal body weights were significantly reduced at 1000 and 5000 ppm, female fetal weights were only significantly reduced at 5000 ppm. There was a statistically significant increase in per litter incidence of reduced ossification of sternebrae 1-4 in the 5000 ppm group. However, this increase was likely related to the fetal growth retardation at this exposure.

Key result

Dose descriptor:

NOAEC

Effect level:

200 ppm

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEC

Effect level:

1 000 ppm

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Significant results of rat developmental toxicity study

Concentration	0 ppm	200 ppm	1000 ppm	5000 ppm
Mean body weight virgins - day of sacrifice (g)	287.6 ± 23.2	290.9 ± 29.0	282.4 ± 29.6	252.7 ± 33.7
Mean body weight dams - gestation day 13 (g)	331.2 ± 20.6	326.4 ± 25.6	325.1 ± 24.1	310.2 ± 33.1
Mean body weight dams - gestation day 20 (g)	408.1 ± 29.2	394.8 ± 34.8	385.2 ± 28.4	366.9 ± 45.0
Mean uterine weight (g)	79.2 ± 14.6	78.2 ± 13.7	73.3 ± 16.5	69.5 ± 13.9
Extra-gestational gain (g)	51.0 ± 13.4	40.8 ± 20.8	39.2 ± 19.8	28.5 ± 17.5
Average fetal weight (g)	3.48 ± 0.37	3.54 ± 0.36	3.27 ± 0.32	2.97 ± 0.38
Average placental weight (g)	0.44 ± 0.05	0.42 ± 0.05	0.41 ± 0.06	0.38 ± 0.05
Average male fetal weight (g)	3.60 ± 0.39	3.66 ± 0.39	3.33 ± 0.33	3.05 ± 0.41
Average female fetal weight (g)	3.33 ± 0.37	3.43 ± 0.37	3.23 ± 0.32	2.86 ± 0.36
Average male placental weight (g)	0.45 ± 0.05	0.43 ± 0.05	0.41 ± 0.05	0.37 ± 0.05
Average female placental weight (g)	0.43 ± 0.05	0.42 ± 0.05	0.41 ± 0.07	0.37 ± 0.05
Reduced ossificantion of sternebrae 1-4 (%)	13.8 ± 21.6	16.3 ± 16.5	29.0 ± 28.6	38.7 ± 23.7

Conclusions:

The LOAEC for maternal toxicity was 1000 ppm based on reduced body weight gain. The NOAEC was 200 ppm for maternal toxicity. The NOAEC for developmental toxicity was 200 ppm (704 mg/m3), and the LOAEC was 1000 ppm (3520 mg/m3) based on reduced fetal weight gain.

Executive summary:

This study examined the effect of inhalation of n-hexane vapors to fetal development. Groups of 30 pregnant and 10 virgin female rats were exposed to concentrations of 0, 200, 1000, or 5000 ppm of test substance vapors for 20 hrs/day, daily, during gestational days 6 -20. During exposure, females were weighed regularly. At the end of the exposure period, the females were sacrificed, and the uterine contents were examined for parameters including number of live fetuses, number of implantations, and number of resorptions. Fetuses were also examined for malformations. Body weight gain was significantly reduced in 1000 ppm exposure females, and body weight was reduced in 5000 ppm exposure females. Fetal weights were also reduced in the 1000 and 5000 ppm exposure groups. A significant increase in reduced ossification of sternebrae 1 -4 in the 5000 ppm fetuses, but this was likely due to the fetal growth retardation. The NOAEC for maternal toxicity is 200 ppm, and the LOAEC is 1000 ppm based on reduced body weight gain. The NOAEC for developmental toxicity is 200 ppm (704 mg/m³), and the LOAEC is 1000 ppm (3520 mg/m³) based on reduced fetal weight gain.

Reference 3

Endpoint:

developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Species:

rabbit

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 830 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

1 substance specific supporting study available for assessment

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

704 mg/m³

Study duration:

subacute

Species:

mouse

Quality of whole database:

2 key and 1 supporting substance specific study available for assessment.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity data is available for n-hexane.

 

Oral

 

n-hexane

A supporting study (Marks et al., 1980) examined the effects of oral exposure of pregnant female mice to the test substance n-hexane. Groups of 6 -34 pregnant female mice were exposed by oral gavage to doses of 0.26, 0.66, 1.32, or 2.20 g/kg once daily, and other groups were exposed three times daily for total doses of 2.17, 2.83, 7.92, or 9.90 g/kg. Dams were exposed during days 6 -15 of gestation and sacrificed on gestation day 18. After sacrifice, the contents of the uterus were examined for number of implants, resorptions, and live and dead fetuses. Fetuses were examined for weight, sex, and malformations. Mortality and reduced weight gain were seen in dams at the 2.20 g/kg dose level and above. Reduced weight gain was seen in fetuses at the 7.92 g/kg dose level and above. No increase in fetal malformations was seen at any dose level. The NOAEL for maternal toxicity was 2170 mg/kg, and the LOAEL was 2200 mg/kg. The NOAEL for developmental toxicity was 2830 mg/kg, and the LOAEL was 7920 mg/kg. The test substance was not teratogenic.

 

Inhalation

 

n-hexane

In a key developmental toxicity study the effect of inhalation of n-hexane vapors to fetal development was examined (Mast, 1987;). Groups of 30 pregnant and 10 virgin female rats were exposed to concentrations of 0, 200, 1000, or 5000 ppm of test substance vapors for 20 hrs/day, daily, during gestational days 6 -20. The LOAEC for maternal toxicity was 1000 ppm based on reduced body weight gain. The NOAEC was 200 ppm for maternal toxicity. The NOAEC for developmental toxicity was 200 ppm (704 mg/m³), and the LOAEC was 1000 ppm (3520 mg/m³) based on reduced fetal weight gain.

 

In another key development toxicity study the effect of inhalation of n-hexane vapors to fetal development was determined (Mast, 1988). Groups of 35 pregnant and 10 virgin female mice were exposed to concentrations of 0, 200, 1000, or 5000 ppm of test substance vapors for 20 hrs/day, daily, during gestational days 6 -17. The maternal NOAEC was 1000 ppm, and the maternal LOAEC was 5000 ppm based on reduced body weight. The developmental LOAEC was 200 ppm (704 mg/m³) based on increased resorptions and reduced uterine weight. There was no NOAEC for developmental toxicity.

 

A supporting study (Bus et al., 1979) was conducted to determine the inhalation developmental toxicity of n-hexane. Groups of pregnant female rats were exposed 1000 ppm of n-hexane vapours for 6 hrs days during gestational days 8-12, days 12-16, or days 8-16. Animals were sacrificed on gestational day 22 and the contents of the uterus examined, and the foetuses examined for abnormalities. One group of animals was allowed to deliver and the growth of the pups examined for the next 7 weeks. There were no abnormalities in the foetuses, therefore the test substance is not teratogenic. There was reduced body weight in pups during the first 7 weeks of life, therefore the LOAEC for developmental toxicity is 1000 ppm (3250 mg/m³).

Justification for classification or non-classification

Based on the available data, n-hexane meets the criteria for classification as a reproductive toxicant (Repr. 2; H361; suspected of damaging fertility or the unborn child) under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).