Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No studies are available for the registered substance 2,2 -Dimethylbutane. Based on the read-across approach, information on n-hexane is used.

In a key reproduction toxicity study the effect of inhalation of commercial hexane (52% n-hexane) on reproduction in rats was determined. The toxicity to reproduction 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).  

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
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.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
reference to same study
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: Systemic toxicity
Key result
Dose descriptor:
LOAEC
Effect level:
9 000 ppm
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
9 000 ppm
Sex:
male/female
Basis for effect level:
other: reproductive toxicity
Key result
Critical effects observed:
no
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 other: ppm (10560 mg/m3)
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
LOAEC
Generation:
F1
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
3 000 other: ppm (10560 mg/m3)
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
LOAEC
Generation:
F2
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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).

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
reference to same study
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: Systemic toxicity
Key result
Dose descriptor:
LOAEC
Effect level:
9 000 ppm
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
9 000 ppm
Sex:
male/female
Basis for effect level:
other: reproductive toxicity
Key result
Critical effects observed:
no
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 other: ppm (10560 mg/m3)
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
LOAEC
Generation:
F1
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
3 000 other: ppm (10560 mg/m3)
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
LOAEC
Generation:
F2
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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).

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:
NOAEC
31 680 mg/m³
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no data available for 2,2 -Dimethylbutane. Data is available from a structural analogue n-Hexane and used as read across.

In a key reproduction toxicity study the effect of inhalation of commercial hexane (52% n-hexane) on reproduction in rats was determined (Daughtrey, 1994; Klimisch score = 1). 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

There is no data available for 2,2 -Dimethylbutane. Data is available from a structural analogue n-Hexane and used as read across.

Two key developmental studies were read across from commercial hexane. In the developmental toxicity study in mice, the maternal NOAEC was 900 ppm, and

the maternal LOAEC was 3000 ppm (10560 mg/m3) based on color changes in the lungs.  The developmental NOAEC was 3000 ppm and the LOAEC was 9000 ppm (31680 mg/m3) in mice.  

In rats, the maternal NOAEC was 3000 ppm (10560 mg/m3), and the maternal LOAEC was 9000 ppm (31680 mg/m3) based on color changes in the lungs, reduced body weight gain, and reduced food consumption. The developmental NOAEC 9000 ppm (31680 mg/m3) in rats.  

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
198903-27 to 1989-04-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because it is well documented and follows OECD Guideline 414.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 42 days at arrival
- Weight at study initiation: 30 g male, 24 g females
- Housing: individually in stainless steel wire mesh cages, identified with toe clips and ear notches
- Diet (e.g. ad libitum): Prolab Certified Rodent Food, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-72 degree F
- Humidity (%): 50-71
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: From: April 5, 1989 To: April 18, 1989
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4320 l glass and stainless steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: 1000 l/min
- Method of conditioning air: Test substance was metered from a piston pump into one or two heated glass evaporator with a temperature of 27-70 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: 1000 l/min
- Air change rate: 20 min, 14 air changes per hour
- Treatment of exhaust air: filtration


TEST ATMOSPHERE
- Brief description of analytical method used: GC with flame ionization detection
- Samples taken from breathing zone: yes, 7 times per exposure
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken seven times per exposure period and analyzed with GC-FID. Distribution of test substance was evaluated by sampling five different areas of the exposure chamber.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: March 27, 1989-April 2, 1989
- Proof of pregnancy: vaginal plug referred to as day 0
Duration of treatment / exposure:
gestation day (GD) 6-15
Frequency of treatment:
6 hrs/day
Duration of test:
GD 18
Remarks:
Doses / Concentrations:
0, 900, 3000, 9000 ppm
Basis: nominal conc.
Remarks:
Doses / Concentrations:
914, 3026, 9107 ppm
Basis: analytical conc.
No. of animals per sex per dose:
30 pregnant females per exposure group
Control animals:
yes
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, clinical signs



BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 15


FOOD CONSUMPTION: Yes

WATER CONSUMPTION: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 18
- Organs examined: gravid uterus, ovaries, cervix, vagina, abdominal cavities, thoracic cavities, liver, kidneys


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter were examined for thoracic and abdominal visceral abnormalities
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
Statistics:
Quantitative continuous variables were compared by use of Levene's test for equal variance, analysis of variance, and t-tests with Bonferroni probabilities. Nonparametric data was evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney U test. Indices were compared using Fisher's exact test. 0.05 was used as the criteria for statistical significance.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no significant treatment related effects to body weight, clinical signs, food consumption, weight changes, or organ weights. There was increased water consumption on GD 6-9, 9-12, 6-15, and 15-18 in the 3000 ppm group. There was also increased water consumption in the 900 ppm group on GD 3-6 and 6-9. There was a statistically significant increase in lung color changes in the 9000 ppm group. Four dams also had brown foci. Two dams in the 3000 ppm group had lung color changes as well, and three had dark brown foci.
Key result
Dose descriptor:
NOAEC
Effect level:
900 ppm
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEC
Effect level:
3 000 other: ppm (10560 mg/m3)
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Colour changes in lungs
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Gestational parameters were similar between exposure and control groups. There was a statistically significant increase in two skeletal malformations in the 9000 ppm group, bilateral bone island at the first lumbar arch, all intermediate phalanges of the hindlimb unossified. No other dose related abnormalities were noted.
Key result
Dose descriptor:
NOAEC
Effect level:
3 000 other: ppm (10560 mg/m3)
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Dose descriptor:
LOAEC
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: vertebra
skeletal: hindlimb
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
9 000 ppm
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Results of Developmental Toxicity Study on Mice

0.0 ppm

900.0 ppm

3000.0 ppm

9000.0 ppm

No. of dams with lung color change

0

0

2

12

All inter. Phalanges (hindlimb) unossified (litters, %)

76.9

72.0

84.0

100.0

Bone island - first lumbar arch - bilateral  (litters, %)

0.0

0.0

8.0

23.1

Conclusions:
In mice, the maternal NOAEC was 900 ppm, and the maternal LOAEC was 3000 ppm (10560 mg/m3) based on color changes in the lungs. The developmental NOAEC was 3000 ppm and the LOAEC was 9000 ppm (31680 mg/m3) in mice.
Executive summary:

The purpose of this study was to examine the developmental toxicity of commercial hexane in mice. Groups of 30 pregnant female mice were exposed to concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day during gestational days 6 -15. The animals were then sacrificed on GD 18. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations.

Necropsy revealed color changes in the lungs of females in the 3000 and 9000 ppm groups. Fetuses in from dams in the 9000 ppm group had a statistically significant increase in some skeletal abnormalities. The maternal NOAEC in mice was 900 ppm (3168 mg/m3), and the LOAEC 3000 ppm based on lung color changes. The developmental NOAEC in mice was 3000 ppm (10560 mg/m3) and the LOAEC 9000 ppm (31680 mg/m3) based on skeletal abnormalities.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-03-24 to 1989-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because it is well documented and follows OECD Guideline 414.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 63 days males, 56 days females at arrival
- Weight at study initiation: 250-300 g male, 175-200 g females
- Housing: individually in stainless steel wire mesh cages, identified with ear tags
- Diet (e.g. ad libitum): Prolab Certified Rodent Food, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 63-74 degree F
- Humidity (%): 40-71
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: From: April 9, 1989 To: April 21, 1989
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4320 l glass and stainless steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: 1000 l/min
- Method of conditioning air: Test substance was metered from a piston pump into one or two heated glass evaporators with a temperature of 27-70 degree C. Conditioned air was passed through the evaporators, where it carried the vapor into the exposure chamber.
- Temperature, humidity: monitored every 30 minutes
- Air flow rate: 1000 l/min
- Air change rate: 20 min, 14 air changes per hour
- Treatment of exhaust air: filtration


TEST ATMOSPHERE
- Brief description of analytical method used: GC with flame ionization detection
- Samples taken from breathing zone: yes, 7 times per exposure
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken seven times per exposure period and analyzed with GC-FID. Distribution of test substance was evaluated by sampling five different areas of the exposure chamber.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: April 2, 1989-April 6, 1989
- Proof of pregnancy: vaginal plug referred to as day 0
Duration of treatment / exposure:
gestation day (GD) 6-15
Frequency of treatment:
6 hrs/day
Duration of test:
GD 21
Remarks:
Doses / Concentrations:
0, 900, 3000, 9000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
914, 3026, 9107 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
25 pregnant females per exposure group
Control animals:
yes
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 15, 18, 21

FOOD CONSUMPTION: Yes

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: gravid uterus, ovaries, cervix, vagina, abdominal cavities, thoracic cavities, liver, kidneys


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter were examined for thoracic and abdominal visceral abnormalities
- Skeletal examinations: Yes: half per litter
Statistics:
Quantitative continuous variables were compared by use of Levene's test for equal variance, analysis of variance, and t-tests with Bonferroni probabilities. Nonparametric data was evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney U test. Indices were compared using Fisher's exact test. 0.05 was used as the criteria for statistical significance.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no treatment related effects to mortality or pregnancy rates. In the 9000 ppm exposure group, there was a significant reduction in weight gain on GD 6-9, and GD 6-15, and slightly reduced on GD 9-12. In the 3000 ppm group, there was a significant reduction in weight gain on GD 9-12, but significantly increased on GD 18-21. Food consumption was significantly reduced in the 9000 ppm group on days 6-9, 9-12, 12-15, and 6-15. There were color changes in the lungs of females in the 9000 ppm group. These changes were also seen in one female each in the 0, 900 and 3000 ppm groups. The color changes in the 900 and 3000 ppm groups were not considered treatment related. There were no treatment related effects to gestational parameters.
Key result
Dose descriptor:
NOAEC
Effect level:
3 000 other: ppm (10560 mg/m3)
Basis for effect level:
other: Maternal systemic toxicity
Key result
Dose descriptor:
LOAEC
Effect level:
9 000 other: ppm (31680 mg/m3)
Basis for effect level:
other: Maternal systemic toxicity
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Reduced body weight gain, reduced food consumption, and colour changes in lungs
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment related effects to the development of fetuses.
Key result
Dose descriptor:
NOAEC
Effect level:
9 000 other: ppm (31680 mg/m3)
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Gestational Body Weight Changes (g)

0.0 ppm

900.0 ppm

3000.0 ppm

9000.0 ppm

Day 0-6

 28.21 (11.819)

31.55 (6.463)

31.19 (9.344)

29.36 (8.245)

Day 6-9

11.55 (8.103)

9.12 (6.009)

11.18 (4.539)

4.34 (6.029)

Day 9-12

16.02 (5.296)

16.31 (5.531)

11.33 (8.446)

12.65 (4.974)

Day 12-15

17.03 (6.807)

19.04 (4.473)

21.84 (9.577)

19.03 (6.453)

Day 15-18

43.52 (9.483)

41.27 (5.755)

37.83 (16.192)

44.11 (9.902)

Day 18-21

51.61 (15.190)

57.79 (8.681)

63.34 (11.295)

57.30 (12.247)

Day 6-15

44.59 (12.727)

44.47 (9.565)

44.35 (9.870)

36.02 (7.850)

Conclusions:
In rats, the maternal NOAEC was 3000 ppm (10560 mg/m3), and the maternal LOAEC was 9000 ppm (31680 mg/m3) based on color changes in the lungs, reduced body weight gain, and reduced food consumption. The developmental NOAEC 9000 ppm (31680 mg/m3) in rats.
Executive summary:

The purpose of this study was to examine the developmental toxicity of commercial hexane in mice. Groups of 25 pregnant female mice were exposed to concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day during gestational days 6 -15. The animals were then sacrificed on GD 21. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations.

Necropsy revealed color changes in the lungs of females in the 9000 ppm groups along with reduced body weight gain, and reduced food consumption. No treatment related abnormalities was seen in the fetuses. The maternal NOAEC in rats was 3000 ppm (10560 mg/m3), and the LOAEC 9000 ppm based on lung color changes, reduced body weight gain, and reduced food consumption. The developmental NOAEC in rats was 9000 ppm (31680 mg/m3).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
10 560 mg/m³
Study duration:
subacute
Species:
mouse
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no data available for 2,2 -Dimethylbutane. Data is available from a structural analogue n-Hexane and used as read across.

In a key developmental toxicity study of commercial hexane (52% n-hexane), groups of 30 pregnant female mice were exposed to concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day during gestational days 6 -15 (API, 1989b; Klimisch score =1). The animals were then sacrificed on GD 18. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations. Necropsy revealed color changes in the lungs of females in the 3000 and 9000 ppm groups. Fetuses in from dams in the 9000 ppm group had a statistically significant increase in some skeletal abnormalities. The maternal NOAEC in mice was 900 ppm (3168 mg/m3), and the LOAEC 3000 ppm based on lung color changes. The developmental NOAEC in mice was 3000 ppm (10560 mg/m3) and the LOAEC 9000 ppm (31680 mg/m3) based on skeletal abnormalities.

In the developmental toxicity of commercial hexane (52% n-hexane) in rats, groups of 25 pregnant female rats were exposed to concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day during gestational days 6 -15 (API, 1989c; Klimisch score = 2). The animals were then sacrificed on GD 21. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations. Necropsy revealed color changes in the lungs of females in the 9000 ppm groups along with reduced body weight gain, and reduced food consumption. No treatment related abnormalities was seen in the fetuses. The maternal NOAEC in rats was 3000 ppm (10560 mg/m3), and the LOAEC 9000 ppm based on lung color changes, reduced body weight gain, and reduced food consumption. The developmental NOAEC in rats was 9000 ppm (31680 mg/m3).

Justification for classification or non-classification

There is no data available for 2,2 -Dimethylbutane. Data is available from a structural analogue n-Hexane and used as read across. Based on available read across data, 2,2 -Dimethylbutane does not warrant classification for reproductive developmental toxicity under the Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP). This is based on the study with commercial n-hexane (52% n-hexane) in which no reproductive or developmental effects were observed.

Additional information