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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
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 restrictions because it was conducted in accordance with testing guideline OECD 416 and is GLP compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

Administration / exposure

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: 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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: reduced body weight
Remarks on result:
other: Generation: F1, F2 (migrated information)
Dose descriptor:
LOAEL
Effect level:
9 000 ppm
Sex:
male/female
Basis for effect level:
other: reduced body weight
Remarks on result:
other: Generation: F1, F2 (migrated information)
Dose descriptor:
NOAEL
Effect level:
9 000 ppm
Sex:
male/female
Remarks on result:
other: Generation: reproductive toxicity (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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)

Applicant's summary and conclusion

Conclusions:
The NOAEL for both male and female rats (adults and offspring) was 3000 ppm (10560 mg/m3). The LOAEL for these groups was 9000 ppm based on reduced body weight. There were no adverse effects to reproduction, therefore the NOAEL 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 NOAEL is therefore 3000 ppm (10560 mg/m3), and the LOAEL is 9000 ppm (31680 mg/m3). Since there were no adverse effects in offspring without adverse maternal effects, the NOAEL for reproduction is 9000 ppm (31680 mg/m3).