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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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.
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
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
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.
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.
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
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: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed
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.
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
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
Reproductive effects observed:
not specified

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

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).

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
2 261 mg/m³
Species:
rat
Quality of whole database:
The available key and supporting studies provide information that is adequate for the purpose of hazard assessment
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The effect of toluene exposures on fertility was reviewed and reported in the EU RAR (2003). No additional relevant data have been found in the updated literature review.

Non-human information

Two key studies are identified.

Roberts et al (2003) conducted a combined two-generation fertility and teratogenicity inhalation study in rats. Exposure to toluene was at 0, 100, 500 or 2000 ppm 6 h/day, 7 days/week during an 80 day pre-mating period and 15 day mating period. Females were further exposed during gestation (GD 1-20) and lactation (LD 5-21). Toluene exposure did not induce adverse effects on fertility and the NOAEC for effects on fertility was 2000 ppm (7500 mg/m3), the highest dose tested.

In the study of Ono et al (1996) rats were exposed to toluene vapour at 600 and 2000 ppm for 6 h/day, and effects on their fertility were investigated. Females were exposed from 14 days before mating until day 7 of gestation. Males were exposed for a total of 90 days, including the mating period; treatment was begun 60 days before pairing, and toxicity with respect to testicular and reproductive functions was examined. Although no abnormalities were seen in mating behaviour or fertility in the males exposed to 2000 ppm decreases in the weights of the epididymides and sperm count were observed. The NOAEC for effects on male fertility was 600 ppm (2261 mg/m3).

Human information

The EU RAR (2003) concluded “In humans, no studies of effects of toluene on sperm count were found. Limited data in humans have not shown indication of effects on fertility in men or menstrual function in women”.  Consequently a value of 2261 mg/m3 from the Ono study will be taken into consideration for risk characterisation.


Short description of key information:
Although no functional effects were seen on fertility in a 2-generation study in rats lower epididymides weight and sperm count were observed at 2000 ppm (7537 mg/m3) with a NOAEC of 600 ppm (2261 mg/m3).

Justification for selection of Effect on fertility via inhalation route:
The available data indicate no functional effect on fertility in a rat 2-generation study, however epididymal weight and sperm count were decreased at 2000 ppm (7537 mg/m3)

Effects on developmental toxicity

Description of key information
Animal studies indicate that toluene is not teratogenic however there is evidence of developmental toxicity (lower birth weight, delayed vaginal opening) at exposures = 1000 ppm in the presence of slight maternal toxicity; the NOAEC for developmental and maternal effects is 600 ppm (2261 mg/m3). Findings suggestive of an increased risk of late spontaneous abortions associated with exposure to toluene at levels around 88 ppm (330 mg/m3) were considered by the Commission Group of Specialised Experts in the fields of Carcinogenicity, Mutagenicity and Reprotoxicity to require confirmation, while the EU Scientific Committee on Occupational Exposure Limits SCOEL (2001) noted that abortions have not been reported upon accidental high exposure or toluene abuse by pregnant women.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non-guideline animal experimental study, published in peer reviewed literature, limitations in design but contributing to a weight of evidence.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Pregnant rats exposed to toluene by whole body inhalation from day 9-21 of pregnancy. Rats were allowed to litter and offspring assessed for range of developmental effects including learning ability and fertility.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Wistar (Bor: Wisw/spf, TNO)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann (Borchen, Germany)
- Age at study initiation: not reported
- Weight on arrival: 180 to 270 g
- Housing: after mating, the females were kept individually caged
- Diet: standard pellet feed (Altromin 1324; Altromin, Lage, Germany), ad libitum
- Water: ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 1 °C
- Humidity: 50 ± 5%
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: not reported
Route of administration:
inhalation: vapour
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1m^3 inhalation chamber and an aerosol-generating system (compressor, pump with calibrated syringe, nozzle). The inhalation conditions were controlled by a Multi-Function Unit (TSE), registering temperature (°C), humidity (%), pressure (mBar), oxygen (%) and flow rate (m^3/h) throughout the exposure period. Controls were placed in the same chamber and inhaled fresh air only.

Samples of the gas mixture in the chamber were taken twice daily and the concentrations were determined by headspace gas chromatography.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Achieved exposure concentrations were within 10% of target
Details on mating procedure:
One male was mated with three female animals from 6:30 to 9:00 a.m.; the following 24 h was designated day 0 of pregnancy if sperm were detected in the vaginal smears. After mating, the females were kept individually caged and were allowed to deliver to term.
Duration of treatment / exposure:
6h/day
Frequency of treatment:
day 9-21 of pregnancy
Duration of test:
Day 0 of pregnancy to delivery and then approximately 90 days after delivery
No. of animals per sex per dose:
38, 23, 23, 29 and 24 for the 0, 300, 600, 1000 and 1200 ppm groups respectively
Control animals:
yes, sham-exposed
Details on study design:
Measurement of toluene blood concentrations: Blood samples were taken from the tail vein on day 20 of pregnancy immediately at the end of exposure from the 300 and 1200 ppm groups. The concentrations of toluene in blood plasma were measured by headspace gas chromatography.
Maternal examinations:
CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes (no details recorded)

POST-MORTEM EXAMINATIONS: No data
Fetal examinations:
As soon as possible after birth the number of viable and dead newborn were counted in each litter, the litter weight was measured and the mean pup weight per litter was calculated.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Toluene exposures of 1000 and 1200 ppm resulted in a reduced body weight of rat dams
Dose descriptor:
NOAEC
Effect level:
1 200 ppm
Basis for effect level:
other: other:
Dose descriptor:
NOAEC
Effect level:
4 522 mg/m³ air (nominal)
Basis for effect level:
other: other:
Dose descriptor:
NOAEC
Effect level:
600 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
2 261 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Toluene exposures of 1200 ppm resulted in lower body weight of offspring at birth and on day 7 and a higher mortality until weaning. Incisor eruption and vaginal opening were also delayed. At 1000 ppm body weight at birth was low and vaginal opening was delayed.
Dose descriptor:
NOAEC
Effect level:
600 ppm
Basis for effect level:
other: Developmental Toxicity
Dose descriptor:
NOAEC
Effect level:
2 261 mg/m³ air (nominal)
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Pregnancy data: The duration of pregnancy was identical in all groups.

 

Litter data: No significant effects on litter size. The mean body weight of offspring of dams exposed to 1000 and 1200 ppm toluene was significantly lower at birth. The reduction of the offspring mean body weight remained until postnatal day 7 in the 1200 ppm group. There was a significantly higher mortality prior to weaning in the 1200 ppm group (7% v. 2.6%).

 

Postnatal development: Incisor eruption and vaginal opening were delayed at 1200 ppm. Vaginal opening was also delayed at 1000 ppm.

 

Fertility: No treatment-related effects.

 

Development of reflexes:There were no differences in the development of reflexes, rota rod performance and locomotor activity between the offspring of animals exposed to toluene and the controls.

 

Locomotor activity:There were no differences in locomotor activity between the offspring of animals exposed to toluene and the controls.

 

Discrimination learning: No concentration-dependent adverse effects were detected.

 

Conclusions:
Toluene exposures up to 1200 ppm do not induce adverse effects on the behaviour of rat offspring exposed during late embryonic and foetal development. The NOAEC for offspring behaviour is 1200 ppm (4522 mg/m3). The NOAECs for maternal toxicity and developmental toxicity are 600 ppm (2261 mg/m3).
Executive summary:

Developmental toxicity and post-natal development were assessed in groups of >20 pregnant female rats exposed to toluene via inhalation 6 h/day during gestation (days 9 -21). Exposure concentrations were 0, 300, 600, 1000 or 1200 ppm. A detailed evaluation of physical development of offspring was conducted and the following tests performed: fore-limb grasp reflex, righting reflex, cliff-drop aversion reflex, maintenance of balance on a rotating rod, locomotor activity, learning ability in a discrimination learning test.

Maternal toluene exposures of 1200 ppm resulted in a reduced body weight gain of rat dams and offspring had lower body weight at birth and day 7 and a higher mortality until weaning. Some developmental landmarks (incisor eruption and vaginal opening) were delayed in this group and are likely to be secondary to the effect on body weight. At 1000 ppm maternal body weight gain was low and offspring had low body weight at birth and delayed vaginal opening. 600 ppm (2261 mg/m3) is the NOAEC for both maternal toxicity and developmental toxicity.

There were no differences in the development of reflexes, rota rod performance and locomotor activity that were attributable to treatment with toluene and no effects were found on learning ability in the operant conditioning task. There were no differences in mating, fertility and pregnancy indexes in the F1-generation. 1200 ppm (4522 mg/m3) is the NOAEC for effects on offspring behaviour.

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
2 261 mg/m³
Species:
rat
Quality of whole database:
The available animal and human data provide information that is adequate for the purpose of hazard assessment
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

LOA is currently reviewing the human and animal data supporting Human Health for Toluene. It is expected to be completed by Q4 2020.

The developmental toxicity of toluene exposures was reviewed and reported in the EU RAR (2003). One additional study published after completion of the RAR has been included in this dossier.

Non-human information

Developmental toxicity has been assessed in a number of studies in several species and there is no evidence that toluene produces malformations in rats or rabbits (Roberts et al, 2007; Saillenfait et al, 2007; Klimisch et al, 1992). However, some evidence of developmental toxicity in the absence of maternal toxicity has been reported in rat inhalation studies (Thiel and Chahoud, 1997; Hass et al, 1999). Roberts and co-workers used whole body inhalation (6 h/day) in pregnant rats exposed to toluene at concentrations of 0, 250, 750, 1500 or 3000 ppm from GD6-15 (Roberts et al, 2007). Toluene induced evidence of maternal toxicity at 3000 and 1500 ppm. There were no adverse effects on implantation number, foetal viability or foetal sex distribution at caesarean section on GD20. Litter weight and mean foetal weight was reduced at 3000 ppm and mean foetal weight was lower at 1500 ppm. Instances of reduced or unossified skeletal elements occurred at the same dose levels. 

The developmental toxicity of toluene was evaluated in the Himalayan rabbit (Klimisch et al, 1992). Pregnant females were exposed to toluene by inhalation 6 h/day at concentrations of 0, 30, 100, 100, 300 or 500 ppm. Toluene was not embryotoxic, foetotoxic, or teratogenic to rabbits.

Thiel and Chahoud (1997) assessed pregnant female rats exposed to toluene via inhalation 6 h/day during gestation (days 9 -21) at concentrations of 0, 300, 600, 1000 or 1200 ppm. A detailed evaluation of physical development of offspring was conducted and included a number of reflex tests, measures of locomotor activity/co-ordination and a discrimination learning test. Maternal toluene exposures of 1200 ppm resulted in a reduced body weight gain of rat dams and offspring had lower body weight at birth and day 7 and a higher mortality until weaning. Some developmental landmarks (incisor eruption and vaginal opening) were delayed in this group and are likely to be secondary to the effect on body weight. At 1000 ppm maternal body weight gain was low and offspring had low body weight at birth and delayed vaginal opening. There were no differences in the development of reflexes, rota rod performance and locomotor activity that were attributable to treatment with toluene and no effects were found on learning ability in the operant conditioning task.

In a study of broadly similar design but using only two exposure levels, groups of pregnant female rats were exposed (gestation days 6-20) to toluene vapour at exposure concentrations of 0, 500 or 1500 ppm (Saillenfait et al., 2007). Maternal toluene exposures of 1500 ppm resulted in a significant (-40%) reduction in corrected maternal body weight gain and a non-significant increase in visceral malformations. The malformations were not fully characterised (described as "mainly diaphragmatic hernia") and affected a total of two foetuses from two litters. In contrast, maternal and foetal parameters at 500 ppm were unremarkable. The LOAEC for maternal and developmental toxicity from this was therefore 1500 ppm, and the NOAEC was 500 ppm.

Rats were exposed to 1200 ppm or 0 ppm toluene for 6 h/day from day 7 of pregnancy until day 18 post-natally (Hass et al, 1999). Developmental and neurobehavioural effects in the offspring were investigated using a test battery including assessment of functions similar to those in the OECD 426, i.e., physical development, reflex ontogeny, motor function, motor activity, sensory function, and learning and memory. The exposure did not cause maternal toxicity or decreased viability of the offspring, however pup birth weight was lower and effects were reported on water maze performance and open field activity. Evaluation of the toxicological relevance of these findings during the EU risk assessment of toluene lead the UK CA to state that there were design limitations as only one exposure level was investigated and it considered that "it is not possible to assess the presence or absence of consistent dose-response relationships". They concluded that there was "weak evidence that toluene may elicit developmental neurotoxicity, manifested as differences in water maze performance and open field activity" and that "the conclusion that toluene is a developmental neurotoxicant to be a tentative one with uncertainties remaining to be addressed". Consequently no conclusion on developmental neurotoxicity can be made on the basis of these data.

It is concluded that toluene is not teratogenic but there is evidence of developmental toxicity (lower birth weight, delayed vaginal opening) at toluene exposure concentrations = 1000 ppm. There are contradicting data as to whether these concentrations are also maternally toxic. In the study of Thiel and Chahoud, maternal body weight at 1000 ppm was statistically significantly lower than concurrent control group (9%) at the end of gestation whereas there were no effects on maternal body weight at 1200 ppm in the study of Hass et al (1999). The NOAEC for developmental effects is 600 ppm (2261 mg/m3) on the basis of lower birth weight and delayed vaginal opening.

The EU RAR (2003) concluded that rat inhalation studies provide strong evidence of developmental toxicity (lower birth weight and long-lasting developmental neurotoxicity) in the absence of maternal toxicity. The effective dose levels are around or more than 1,000 ppm (3752 mg/m3). The NOAEC for lower birth weight and delayed postnatal development is set at 600 ppm (2250 mg/m3). The RAR noted that a NOAEC for developmental neurotoxicity cannot be determined from the available studies but the LOAEC for this effect is 1,200 ppm (4500 mg/m3). The RAR also reported that toluene induces similar effects in mice while data in rabbits are insufficient to evaluate the sensitivity of this species compared to rats and mice. Appendix D of the EU RAR documents that not all Member States agreed with the interpretation of the animal developmental neurotoxicity studies.

Human information

In a study designed to examine pregnancy outcomes in women occupationally exposed to toluene (Ng et al., 1992), rates of late spontaneous abortion (12-28 weeks) were compared by retrospective questionnaire in 55 women (105 pregnancies) exposed to toluene (mean 88 ppm, range 50-150 ppm: high exposure group), 31 women (68 pregnancies) working in the same factory in departments where much lower exposure to toluene occurred (range 0-25 ppm: low exposure group), and an external community control group of 190 women (444 pregnancies) attending antenatal and postnatal clinics. The study is assigned Klimisch Rel. 4 (not assignable) due to inconsistencies in its design and reporting/analysis of the results obtained (see below). The rate of late spontaneous abortion for the high exposure group was significantly higher than in the low exposure group (OR 4.80, 95% CI 1.01-22.86). The rate was also higher than for the pregnancies of women attending antenatal and postnatal clinics, but it was not stated that the current pregnancies of this group were excluded from the study which is necessary to avoid bias (the most recent pregnancy of a postnatal recruit or an antenatal attendee who was more than 28 weeks pregnant cannot be a late spontaneous abortion). Early spontaneous abortions in the first months after conception were not recorded which may have lead to misclassification, while in the maternal health clinic group there were 27 pregnancies that were neither late spontaneous abortions nor live births, but were not characterised further. Exposure information was obtained from a separate, earlier study of 41 female and 9 male workers at the same factory who performed assembly jobs in a sitting position and who were exposed to relatively steady levels of toluene emitted from glue used for bonding electronic parts (Foo et al., 1988); exposures were in a range 8.5 ppm to 262.7 ppm, with 7 measurements >150 ppm and 10 measurements <50 ppm i.e more variable than information presented by Ng et al. (1992). Furthermore, the exposure study by Foo et al (1988) did not include workers from departments from which the low exposure group was recruited. Overall therefore, this study provides weak evidence of an elevated rate of late spontaneous abortions at 88 ppm.

The findings of Ng et al. were considered by the EU RAR (2003) to suggest “an increased risk of late spontaneous abortions associated with exposure to toluene at levels around 88 ppm (range 50-150 ppm)”, although it was also noted that the study “cannot be used to establish definitively a causal relationship between late spontaneous abortions and toluene exposure or the magnitude of the LOAEL”. Nonetheless, the EU RAR (2003) concluded that exposure of pregnant women to the levels experienced by the High exposure group would raise “serious ethical concerns” based on the current evidence suggesting an increased risk for late spontaneous abortions. The results of this study were also considered by the Commission Group of Specialised Experts in the fields of Carcinogenicity, Mutagenicity and Reprotoxicity (SEG, 2000) who concluded that the findings were of concern but in need of confirmation, and corroborating evidence from experimental studies was lacking. SCOEL (2001) observed that abortions have not been reported upon accidental high exposure or toluene abuse by pregnant women (Wilkins-Haug and Gabow, 1991) and that an exposure limit of 50 ppm (192 mg/m3) would protect against potential foetotoxicity.

A review by Bukowski (2001) examined the epidemiological evidence for adverse reproductive outcomes from occupational studies that presented toluene-specific findings. Clinical investigations of the reproductive effects of toluene abuse were also examined.  The reproductive outcomes of concern in these studies were primarily spontaneous abortion (SA), congenital malformation (CM), or decreased fertility/fecundity. As these were observational research studies not controlled experiments a number of potentially important biases were identified: (1) selection bias, (2) recall bias, and (3) confounding. It was concluded that the literature reviewed cannot be used to definitively establish a causal relationship between toluene exposure and spontaneous abortion or congenital malformation or the magnitude of the lowest effect level.


Justification for selection of Effect on developmental toxicity: via inhalation route:
Animal studies indicate that toluene is not teratogenic however there is evidence of developmental toxicity (lower birth weight, delayed vaginal opening) at exposures = 1000 ppm in the presence of slight maternal toxicity.

Justification for classification or non-classification

There is no evidence that toluene has any effect on fertility and does not warrant classification for reproductive toxicity.

Lower birth weight in the absence of significant maternal toxicity and weak evidence of behavioural effects (water maze and open field performance) was seen in offspring of dams exposed to toluene from day 7 of pregnancy until day 18 post-natally. Toluene is therefore classified as Category 2 (H361d) according to GHS.

Additional information