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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline, non-GLP, animal experimental study, published in peer reviewed literature, notable limitations in design, adequate for assessment.

Data source

Reference
Reference Type:
publication
Title:
Reproductive and developmental toxicity studies of toluene II. Effects of inhalation exposure on fertility in rats
Author:
Ono A, Sekita K, Ogawa Y, Hirose A, Suzuki S, Saito M, Naito K, Kaneko T, Furuya T, Kawashima K, Yasuhara K, Matsumoto K, Tanaka S, Inoue T and Kurokawa Y
Year:
1996
Bibliographic source:
Journal of Environmental Pathology Toxicology and Oncology 15, 9-20

Materials and methods

Principles of method if other than guideline:
Male and female rats were exposed to toluene vapour and effects on their fertility were investigated. Toxicity with respect to testicular and reproductive functions was examined.
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Toluene
EC Number:
203-625-9
EC Name:
Toluene
Cas Number:
108-88-3
Molecular formula:
C7H8
IUPAC Name:
toluene
Details on test material:
- Name of test material (as cited in study report): toluene
- Physical state: liquid at room temperature
- Analytical purity: 98%
- Vapour pressure: 18.38 mmHg at 20°C
- Source: Wako Chemical Co., Tokyo, Japan

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Japan SLC, Inc., Hamamatsu, Japan
- Age at delivery: 6 weeks (males); 9 weeks (females)
- Housing: The male rats were individually housed in wire-bottomed stainless steel cages (25.5 x 22 x 20 cm) installed in exposure chambers, and the female rats were housed, three rats per cage, in polycarbonate cages (37 x 21x 18 cm) bedded with white chips.
- Diet: basal diet, F-2 (Funabashi Farm Co., Japan) ad libitum except during exposure
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 25±2°C
- Humidity: 50±10%
- Air changes: changed continuously
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel, with a volume of 1.56 m3
- Method of holding animals in test chamber: in wire-bottomed stainless steel cages
- Air flow rate: 0.32 m3/min

TEST ATMOSPHERE
- Brief description of analytical method used: the vapour concentration of toluene in the chamber was monitored every 15 min by automated injection of an atmospheric sample into a gas chromatograph
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually in polycarbonate cages
- Any other deviations from standard protocol: 15 animals per group
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The standard deviation of the exposure levels was less than 10% during all exposures
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
7 days/week Male rats were exposed for 90 days (60 days pre-mating, during mating period and until termination on day 91); female rats were exposed from 14 days before mating, during the mating period and until day 7 of gestation
Details on study schedule:
On day 20 of gestation, all pregnant females in each group were anaesthetized with ether, decapitated, and autopsied.
On day 91 after starting exposure, 8 males in each group were anaesthetized with ether, and killed by decapitation. Quantitative morphometry of the spermatogenic cycle stages was carried out. The remaining males in all groups were examined for spermatozoa and elemental analysis of the testis on the next day.
Doses / concentrations
Remarks:
Doses / Concentrations:
600 or 2000 ppm (2261 or 7537 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
15
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on the findings of previous studies.

Examinations

Parental animals: Observations and examinations:
OBSERVATIONS: Yes
- Time schedule: daily, immediately before and after toluene exposure (males and females)

BODY WEIGHT: Yes
- Females: on days 0, 4, 8 and 14 after the start of exposure and daily throughout gestation
- Males: weekly throughout the experimental period except during the mating period

FOOD CONSUMPTION: Yes
- Females: recorded after exposure for 4 and 14 days
- Males: weekly throughout the experimental period except during the mating period
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
Eight males from each group were sacrificed the day after the last exposure. Quantitative morphometry of the spermatogenic cycle stages was carried out.
Litter observations:
Not applicable
Postmortem examinations (parental animals):
- The animals were anesthetized with ether and blood samples were collected from males for haematological and serum biochemical studies through the orbital vein before termination.
- Brain, heart, lung, liver, kidneys, spleen, pituitary, adrenals, thymus, testes and epididymides were weighed.
- All the organs listed above, except the testes, were fixed in 10% buffered formalin solution for routine histological processing. Paraffin sections were stained with haematoxylin and eosin (H & E) for histopathological examination.
- Bone marrow, obtained from the femur by aspiration at autopsy, was examined.
Postmortem examinations (offspring):
Foetuses were removed by Caesarean section. The peritoneal cavity and uterus were opened, and the number of corpora lutea, living foetuses, dead foetuses, and resorptions were counted, along with the implantation sites. All living foetuses removed from the uterus were sexed, weighed, and inspected for external malformations.
Statistics:
Unit of analysis for offspring was the litter. Statistical significance was assessed using the x2, rank-sum and t tests. If the t-test variance was homogeneous, the Student's method was applied, and, if not, the Aspin Welch method. A difference was considered statistically significant at p <0.05 or p <0.01.
Reproductive indices:
Copulation index, fertility index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no mortalities. The high toluene dose caused salivation and lachrymation in all females during daily exposure from the 20th day of exposure and onwards. The effects ceased immediately on removal from exposure.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The number of spermatogenic cells counted at 3 stages was not affected by toluene exposure. The sperm count was reported to be significantly decreased at 2000 ppm although details were not provided. Sperm motility was not affected.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance and fertility were not affected by the toluene exposure. Female rats were paired on a 1:1 basis with male rats of the same dose group. Except for one rat pair in the 600 ppm group, all pairs copulated. Only one female rat, in the 2000 ppm group, did not become pregnant. The copulation index was 100% in the control, 93.3% in the 600 ppm and 100% in the 2000 ppm exposure groups. The fertility indices in the control, 600, and 2000 ppm groups were 100, 100, and 93.3%, respectively.

No statistically significant differences were observed between exposed and unexposed dams with respect to number of corpora lutea, implantations, live foetuses, sex ratio, malformations (0 in all groups), foetal weight, or foetal deaths. In the 2000 ppm group, foetal mortality was higher than in the control group and the number of dams with dead foetuses was increased.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males exposed to 2000 ppm higher kidney weight was accompanied by basophilic changes and tubular necrosis. Thymus weight and relative and absolute epididymides weights were lower at 2000 ppm.

HAEMATOLOGY AND CLINICAL BIOCHEMISTRY
There were no effects on haematology and clinical biochemistry parameters that were related to exposure with toluene

HISTOPATHOLOGY (PARENTAL ANIMALS)
No abnormalities of testes and epididymides were detected on histopathological examination.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Effect level:
600 ppm
Sex:
male
Basis for effect level:
other: based on decreased sperm count and reduced epididymides at 2000 ppm
Dose descriptor:
NOAEC
Effect level:
2 261 mg/m³ air (nominal)
Sex:
male
Basis for effect level:
other: based on decreased sperm count and reduced epididymides at 7537 mg/m3

Results: F1 generation

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
600 ppm
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other:
Remarks:
Based on increased foetal mortality at 2000 ppm
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
2 261 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other:
Remarks:
Based on increased foetal mortality at 2000 ppm

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Caesarean section data of females exposed from day 14 premating to day 7 of pregnancy and killed on day 20 of pregnancy

(Table based on Ono A et al, 1996, Journal of Environmental Pathology Toxicology and Oncology 15, 9-20,Table 2)

 

0 ppm

600 ppm

2000 ppm

Number of dams

15

14

14

Number of corpora lutea

13.3±1.3

14.5±1.1

14.9±1.7

Number of implantations

12.1±1.9

13.0±0.8

13.3±3.5

% to corpora lutea

90.9±10.7

96.2±4.3

88.1±21.0

Number live foetuses

11.1±2.5

13.0±1.5

11.6±3.9

% to implantations

91.2±12.9

93.3±8.6

87.9±16.0

Sex ratio (male/female)

82/84

95/87

86/77

Bodyweight of live foetuses (g)

male

3.59±0.20

3.61±0.19

3.56±0.14

female

3.49±0.19

3.45±0.19

3.46±0.64

Number of foetal deaths

1.0±1.5(7)

0.9±1.2(7)

1.6±1.8(11)

% to implantations

8.8±12.9

6.7±8.6

11.5±14.2

Numbers in parentheses indicates number of dams with dead foetuses

 

Organ weights (relative to final bodyweight) for groups of eight male rats exposed to toluene for 90 days (g%)

(Table based on Ono A et al, 1996, Journal of Environmental Pathology Toxicology and Oncology 15, 9-20,Table 6)

 

0 ppm

600 ppm

2000 ppm

Kidney

0.51±0.04

0.53±0.03

0.59±0.06**

Thymus

0.08±0.02

0.07±0.01

0.06±0.01*

Epididymis

right

0.13±0.01

0.12±0.01

0.11±0.01**

 

left

0.13±0.01

0.12±0.00*

0.11±0.01**

* p<0.05, ** p<0.01

Applicant's summary and conclusion

Conclusions:
Toluene showed no effects on fertility in rats, however, decreased sperm count was reported at 2000 ppm (90 days, 6 h/day). The NOAEC for this effect was 600 ppm (2261 mg/m3).
Executive summary:

The effects of toluene of fertility were examined in male and female Sprague-Dawley rats exposed to vapour at 600 or 2000 ppm for 6 hours/day. Females were exposed from 14 days prior to mating until day 7 of gestation; males were exposed for a total of 90 days (including 60 days pre-mating and during mating).

In females at 2000 ppm, acute clinical signs of salivation and lachrymation were observed during exposures only. Although no abnormalities were seen in mating behaviour or fertility, foetal mortality and the number of dams with dead foetuses was marginally increased in the 2000 ppm group. A number of differences were noted for the males exposed to 2000 ppm compared to control: an increase in kidney weight, a decrease in thymus weight, greater incidence of basophilic changes and necrosis of kidney tubules and decreases in the epididymides weight and sperm count.

The NOAEC for effects on male fertility was 600 ppm (2261 mg/m3).