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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant (assumed), near guideline, animal experimental study, published in peer-reviewed literature, some limitations in design but otherwise adequate for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
exposure GD6-15. Compliant with guideline of the time but not current (2001) version
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
no information reported

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass., USA
- Housing: 5/cage in stainless steel mesh cages
- Diet: Purina Laboratory Chow ad libitum except during exposure
- Water: ad libitum except during exposure

ENVIRONMENTAL CONDITIONS: No details reported

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
Benzene was generated as vapour by bubbling metered house air through a fritted glass tube submerged in benzene in a 500 mL Erlenmeyer flask
with a glass-wool filter plug in the effluent line. The vapour was mixed with filtered conditioned air drawn into the chamber turret at 1000 L/min
via a tangential duct which assured rapid dispersion within the pyramidal top of the chamber. Analytical concentrations of benzene vapour were determined once daily by gas chromatography.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentrations of benzene vapour were determined once daily by gas chromatography. The mean chamber concentrations (±SD) were 9.75±1.16, 52.9±5.33 and 513.9±25.3 ppm for the target concentrations of 10, 50 and 500 ppm respectively.
Details on mating procedure:
Two females were housed with one male until signs of insemination were observed by microscopic examination of the returns from a vaginal douche.
If sperm or a vaginal plug were observed, the female was assumed to have mated and was removed from the breeding regimen. The day on which evidence of mating was observed was designated as Day 0 of gestation.
Duration of treatment / exposure:
gestation days 6-15
Frequency of treatment:
daily, 7 h/day
Duration of test:
up to gestation day 20
No. of animals per sex per dose:
17, 18, 20, 19 rats for the 0, 10, 50 and 500 ppm groups respectively
Control animals:
yes, sham-exposed

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS AND MORTALITY: Yes (mortality, signs of toxicity and pregnancy rate)
- Time schedule: Not reported

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 5, 15 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

HAEMATOLOGY: At gestation Day 5 and just prior to caesarean delivery on Day 20, venous blood samples were obtained from each female and erythrocyte, total leukocyte, and differential leukocyte counts were determined.

Ovaries and uterine content:
On day 20 females were sacrificed, caesarean sections were performed and numbers of ovarian corpora lutea, resorption sites and live and dead foetuses examined.
Fetal examinations:
Each foetus was examined externally and weighed. Whole-body transverse sections were made and examined microscopically for visceral changes (approximately one third of foetuses). The skeletons of the remaining foetuses were examined microscopically for anomalies and ossification.
Statistics:
The variances of the mean changes in maternal body weights and in means of the mean litter foetal body weights and lengths of each treated group were tested for statistical significance (F test). If there were no significant difference among the variances, the differences in means were tested by Student's t test. However, if there were a significant difference among the variances, the differences in means were tested by Cochran's approximation of t . Mean maternal body weights and body weight gains were also evaluated using analysis of covariance and the method of Gomes and Howell. The reproduction indices and the number of foetal variants of the control and treated groups were analyzed by the χ2 method or regression analysis.
All differences that would occur less than 5% of the time by chance were accepted as statistically significant.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
32 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Basis for effect level:
other: Developmental Toxicity
Dose descriptor:
NOAEC
Effect level:
32 mg/m³ air (nominal)
Basis for effect level:
other: Developmental Toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Maternal toxicity

No deaths or signs of toxicity were noted in the dams and the pregnancy rate was within the expected range.

 

 

0 ppm

10 ppm

50 ppm

500 ppm

Number of dams mated

17

18

20

19

Number of dams pregnant

11

15

15

14

Pregnancy rate (%)

64.7

83.3

75.0

73.7

 

Maternal weight at 50 and 500 ppm was significantly lower than control on day 15 and maternal weight gain from days 5 -15 was also reduced at these dose levels:

 

Maternal bodyweight

0 ppm

10 ppm

50 ppm

500 ppm

Day 5:

Mean weight (g)

 

239

 

252

 

251

 

241

Day 15:

Mean weight (g)

Mean gain days 5-15 (g)

 

298

59

 

306

54.2

 

290*

39*

 

278*

37*

Day 20:

Mean weight (g)

Mean gain days 15-20 (g)

 

339

42

 

362

56

 

338

48

 

337

59

* significantly different from control

 

No treatment-related effects were seen in any of the haematology parameters, at necropsy or on the number of implantation sites, resorbed and dead foetuses, incidence of live foetuses or sex distribution of the foetuses.

 

Foetal effects

Statistically significant decreases in the mean crown-rump distance of the high-dosed foetuses and mean body weights of the mid- and high-dosed live foetuses were noted when compared with the controls.

 

Live foetuses

0 ppm

10 ppm

50 ppm

500 ppm

Mean body weight (g)

4.4

4.4

3.8*

3.6*

Mean crown-rump length (cm)

4.1

4.1

3.9

3.8*

* significantly different from control

 

Statistically significant increases in the incidence of skeletal findings indicative of incomplete ossification of the skeleton at 50 and 500 ppm. There was no evidence of treatment-related foetal dysmorphogenesis.

 

Skeletal findings:

0 ppm

10 ppm

50 ppm

500 ppm

Number of litters examined

11

15

15

13

Number of foetuses examined

75

134

91

98

Foetuses with delayed ossification:

In skull

In vertebral column

In rib cage

In pelvic girdle

In extremities

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

17

0

17

 

8

9

23

8

17

Foetuses with variants:

In ribs

In forefeet

 

0

0

 

0

0

 

0

0

 

1

2

Foetuses with anomalies

In skull

0

0

0

1

Mean values:

No. of caudals

No. of metacarpals and phalages

No of metatarsals and phalages

 

5.4

10.5

8.9

 

5.3

11.0

9.2

 

4.6

9.4

7.6

 

4.0*

8.9

7.7

 Table based on Kuna and Kapp, 1981 Table 3

Visceral examination revealed slight dilation of the ventricles in the brain in five mid- and four high-dosed animals but there was no clear evidence for an association with treatment with benzene.

Applicant's summary and conclusion

Conclusions:
Benzene inhalation at 50 ppm (160 mg/m3) during pregnancy induces maternal and developmental toxicity. There was no evidence of teratogenicity at the highest dose tested 500 ppm (1600 mg/m3). The NOAEC for teratogenicity was 500 ppm (1600 mg/m3); the NOAEC for maternal and developmental toxicity was 10 ppm (32 mg/m3).
Executive summary:

Developmental toxicity was investigated in groups of pregnant female rats exposed 7h/day from day 6 to day 15 of gestation to benzene concentrations of 0, 10, 50 or 500 ppm (0, 32, 160, 1600 mg/m3). On gestation day 5 and prior to termination venous blood samples were taken from each female and erythrocyte, total leukocyte and differential leukocyte counts determined. Dams were killed on day 20 and the following observations made: number of corpora lutea, number of resorptions, number of live and dead foetuses. Foetuses were examined for external, skeletal and visceral abnormalities, sex was determined and body weight and crown-rump length measured.

No deaths or signs of toxicity were noted in the dams. Maternal body weight and body weight gain was decreased at 50 and 500 ppm. No treatment-related effects were seen in any of the haematology parameters, necropsy findings or on the number of implantation sites, resorbed and dead foetuses, incidence of live foetuses or sex distribution of the foetuses. Statistically significant decreases in the mean crown-rump length of the 500 ppm group foetuses and mean body weights of the 50 ppm and 500 ppm live foetuses were noted when compared with the controls. As a consequence these foetuses showed signs of incomplete ossification in comparison with the larger and heavier control foetuses. Visceral examination revealed slight dilation of the ventricles in the brain in five 50 ppm and four 500 ppm animals.

The NOAEC for teratogenicity was 500 ppm (1600 mg/m3); the NOAEC for maternal and developmental toxicity was 10 ppm (32 mg/m3).