Hydrocarbons, C5-rich

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Near-guideline, GLP, animal experimental study, published in peer reviewed literature, minor limitations in design, adequate for assessment.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

study pre-dates current guideline (2001) and does not assess sperm parameters or length of oestrous cycles

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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: clear, colourless liquid
- Analytical purity: 99.9%
- Supplier: American Petroleum Institute, Washington, DC, USA

Test animals

Species:

rat

Strain:

other: Crl:CD[SD]BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI, USA
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: not reported
- Housing: individually in wire mesh cages or in plastic breeding cages with nesting material in 16m3 stainless steel and glass exposure chambers with adjoining anterooms. Two additional 16 m3 chambers housed pups during the lactation period.
- Diet (e.g. ad libitum): no details reported
- Water (e.g. ad libitum): no details reported
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 23-24.1°C
- Humidity: 52-56%
- Air changes (per h): not reported
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: not reported

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: housed in 16 m3 exposure chambers throughout study
- System of generation: toluene was metered at controlled rates to a column where it was vaporised with heated nitrogen; concentrated vapours were diluted with chamber ventilation air to produce desired dose levels

TEST ATMOSPHERE
- Brief description of analytical method used: gas phase spectrophotometry using a Miran infrared analyzer
- Samples taken from breathing zone: yes
- Nominal concentrations were determined from weight of test material used divided by total volume of airflow in each chamber during exposure.

Details on mating procedure:

- M/F ratio per cage: 2 females:1male
- Length of cohabitation: maximum of 15 days
- Proof of pregnancy: vaginal lavage. Day on which evidence of copulation was observed was designated day 1 of gestation (GD1)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual exposure concentrations were determined at hourly intervals by gas phase spectrophotometry using a Miran infrared analyser

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

7 days/week

Details on study schedule:

Adults of both the F0 and Fl generations were exposed for 6h/day, 7 days/week during 80 days premating and 15-day mating period. F0 and Fl males were not exposed after completion of the mating period. Presumed pregnant females of both generations were exposed from gestation day (GD)
1-20 and lactation day (LD) 5-21. At day 5 of lactation, females were removed from their litters, exposed for 6 h/day, 7 days/week, and returned to the litters following daily exposure. Parental females were not exposed after litters had been weaned. F1 pups selected to produce the F2 generation
were treated for a minimum of 80 days beginning immediately after weaning, and initially mated at a minimum of 100 days of age. F2 pups were not exposed to toluene by inhalation. Target exposure concentrations were 0, 100, 500 and 2000 ppm (0, 375, 1875 and 7500mg/m) for parental animals of both sexes and 2000 ppm to males or females mated to untreated partners.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 375, 1875, 7500 mg/m3
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 102, 497, 2020 ppm
Basis:
analytical conc.
average over 39 weeks of exposure

No. of animals per sex per dose:

F0 generation (parental): 30, 10, 10, 20, 10, 10 males and 60, 20, 20, 40, 20, 20 females for the 0, 100, 500, 2000, 2000 (females untreated) and 2000 (males untreated) groups respectively.
F1 generation: 19, 10, 10, 10, 10, 10 males and 40, 20, 19, 19, 20, 20 females for the 0, 100, 500, 2000, 2000 (females untreated) and 2000 (males untreated) groups respectively.

Control animals:

yes, sham-exposed

Details on study design:

There were three 2000 ppm groups. In one group males and females were exposed, in the second the females were not exposed but were mated with exposed males, in the third the males were not exposed but were mated with exposed females.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, before and after exposure for signs of overt toxicity, changes in general appearance and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly on F0 parental rats and F1 pups selected for mating

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for adult rats. Mated females were weighed on GD 1, 7, 14, and 21 and on LD 1, 4, 14, and 21

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Parental food consumption was measured weekly from initiation to termination of each generation except during mating, and calculated as group mean food consumption (by sex) in units of g/rat/day and g/kg body weight/day

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Confirmation of oestrus (normal cycling) among adult F0 and Fl females was determined by vaginal lavage daily during mating period

Sperm parameters (parental animals):

Not assessed

Litter observations:

STANDARDISATION OF LITTERS: No

CAESARIAN DATA: On day 21 of gestation, 20 mated F0 females from control and 2000 ppm (both sexes treated) groups were killed, uteri were removed, weighed (with and without foetal content) and foetal contents examined. The following were recorded: number and location of viable and non-viable foetuses, early and late resorptions, number of total implantations and corpora lutea; the number, condition, sex, location and weight of each live and dead foetus, and external alterations and malformations of live foetuses.

REPRODUCTIVE AND LITTER EVALUATIONS: All females not selected for Caesarean section, were put into individual delivery cages and examined twice daily for signs of parturition. The day on which all pups were delivered was designated day 1 of lactation (LD). Parameters recorded included: male and female fertility indices, length of cohabitation (copulatory interval), gestation period, abnormalities in nesting and nursing behaviour, difficulties at parturition, mean number of live and stillborn pups per litter, pup survival through weaning, pup weight at birth and during lactation, and general appearance and behaviour.

PUP BODY WEIGHT: Pups were individually weighed on LD 1, 4, 14, and 21.

PUP CLINICAL SIGNS: Animals were observed twice daily, before and after exposure, for signs of overt toxicity, changes in general appearance and mortality. Detailed observations were recorded weekly on F1 pups selected for mating. Examinations of Fl and F2 pups for gross deformities
were conducted on days 1, 4, 14, and 21 of lactation.

Postmortem examinations (parental animals):

All adult animals dying or killed moribund were necropsied. Reproductive organs and gross lesions were examined from all selected parental animals.

Postmortem examinations (offspring):

One pup/sex/litter from F1 and F2 weanlings (control and 2000 ppm groups) selected for histopathology. Half of F0 and F1 males per group were necropsied with histopathology after the mating period; remaining males were retained untreated and necropsied 3 weeks after the end of the mating period. All F0 and Fl females that delivered pups were necropsied with histopathology at weaning on day 21 of lactation.

The following were examined from selected F1 and F2 weanlings: gross lesions, adrenals, bone marrow, brain, colon, duodenum, eyes, epididymides, heart, ileum, liver, lung, lymph node, mammary gland, ovaries, pancreas, salivary gland, seminal vesicles, skeletal muscle, skin, cervical spinal cord, spleen, stomach, testes, thyroid, urinary bladder, uterus, prostate, and thymus.

Statistics:

Body weight, body weight gain, food consumption, live pups and foetuses, pup and foetal body weight analysed by one-way ANOVA followed by Bartlett's test for homogeneity of variance and appropriate t-test (i.e. equal or unequal variance). Dose-response relationships assessed using simple linear regression. Early/late resorptions, dead foetuses, post-implantation loss and pup survival indices analysed using the Mann-Whitney U-test and Jonckheere's test. Fertility indices, pregnancy rates, malformations, sex ratios analysed using Fisher's exact test and the Armitage test for linear trend in proportions. The litter was the experimental unit.

Results and discussion

Results: P0 (first parental generation)

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

500 ppm

Sex:

male/female

Basis for effect level:

other: slightly lower body weight gain 2000 ppm

2

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

1 875 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: slightly lower bodyweight gain at 7500 mg/m3

3

Dose descriptor:

NOAEC

Remarks:

reproduction

Effect level:

2 000 ppm

Sex:

male/female

Basis for effect level:

other: no effects on fertility, reproductive performance or maternal/ pup behaviours at 2000 ppm (highest dose tested)

4

Dose descriptor:

NOAEC

Remarks:

reproduction

Effect level:

7 500 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: no effects on fertility, reproductive performance or maternal/ pup behaviours at 7500 mg/m3 (highest dose tested)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Effect levels (F1)

1

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

500 ppm

Sex:

male/female

Basis for effect level:

other: lower body weight at birth and body weight gain prior to weaning

2

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

1 875 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: lower body weight at birth and body weight gain prior to weaning

Results: F2 generation

Effect levels (F2)

1

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

500 ppm

Sex:

male/female

Basis for effect level:

other: lower body weight at birth and body weight gain prior to weaning

2

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

1 875 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: lower bodyweight at birth and body weight gain prior to weaning

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Analysis of the chamber atmospheres demonstrated that actual exposures were within 1-2% of nominal.

ADULT OBSERVATIONS
There were no toluene-related deaths in either parental generation. 

Body weight gain was decreased slightly in mid- and high dose F0 males during the first 3 weeks of treatment but was unremarkable thereafter. Similar decrements were present in F1 animals of both sexes throughout the exposure period, with statistically significant effects intermittently present in this instance. Similar decreases, with occasional statistical significance, were also present in females during the lactation period. Representative mean body weight data for females from control and high dose females is summarised below:

(Table based on Roberts L et al 2003, Reproductive Toxicology, 17, 649-658,Table 3)

	F0 females				F1 females
	0	H(a)	H(b)	H(c)	0	H(a)	H(b)	H(c)
GD1	257	255	262	244	269	240	266	238
GD7	283	280	281	265	296	267	288	261
GD14	310	308	311	290	322	294	318	290
GD21	373	367	370	348	389	346	383	347
LD1	297	290	301	277	307	276	298	272
LD4	295	275	299	272	312	285	308	281
LD14	324	312	326	294	329	287	323	290
LD21	321	306	316	296**	318	284**	315	286**
H = high dose (2000 ppm) (a) both sexes treated (b) males only treated (c) females only treated * P , 0.05, ** P < 0.01

Comment: the authors note that F1 females showed a slight but consistent reduction in body weight throughout their life, which appeared to a correlate with reduced growth when they were pups linked to continued lower body weight as they matured into adults.

Maternal behaviour and food intake was comparable over the groups.

Dams from the 2000 ppm caesarean section group had terminal body weights comparable to controls with no significant differences in liver or kidney weights. Mean intact uterine weight was lower in the 2000 ppm females (65.9 g) than controls (69.8 g) reflecting low foetal weights. respectively).

Fertility indices, duration of mating and gestational length were comparable among the groups.

FOETAL OBSERVATIONS
Foetal body weights were significantly lower in the 2000 ppm group relative to the controls (3.3 versus 3.6 g; p<0.01), with more developmental variations present in the treated animals (88 foetuses in 17 litters) than the controls (57 foetuses in 18 litters). The incidence of skeletal variations, rudimentary ribs and unossified sternebrae was increased in the 2000 ppm groups, but considered by the authors to be related to the lower foetal body weight:

	0 ppm	2000 ppm (a)
No. of skeletal examinations	117	112
Rudimentary ribs	17(10)	34(11)
Unossified sternebrae	32(13)	57(14)
Total developmental variations	57(18)	88(17)

Results presented as number of foetuses exhibiting variations (number of litters affected in brackets)
(a) Both sexes treated 
No statistically significant differences

OFFSPRING OBSERVATIONS
Pup viability, mean litter size and lactational survival were comparable among the groups. Body weight gain of high dose group pups of both sexes was significantly less than the controls throughout lactation in both F1 and F2 generations. 

HISTOPATHOLOGY
Microscopic examination of 29 tissues from selected control and high dose animals in the F0, F1 and F2 generations found no treatment-related effects. 

Applicant's summary and conclusion

Conclusions:

The results indicate a reproductive NOAEC of at least 2000 ppm (7500 mg/m3), a parental NOAEC of 500 ppm (1875 mg/m3) based on marginally decreased body weight, an offspring NOAEC of 500 ppm (1875 mg/m3) based on decreased body weight/weight gain and a foetal/developmental NOAEC of 500 ppm (1875 mg/m3) based on decreased foetal weight and increased skeletal variations.

Executive summary:

Reproductive and developmental toxicity of toluene was assessed in a combined two-generation fertility and teratogenicity inhalation study in groups of at least 10 male and 20 female Crl:CD[SD]BR rats. Exposure to toluene was at 0, 100, 500 or 2000 ppm (0, 375, 1875 or 7500 mg/m3) 6 h/day, 7 days/week during an 80 day pre-mating period and 15 day mating period. Females were further exposed on GD 1 -20 and LD 5 -21.

Toluene exposure did not induce adverse effects on fertility, reproductive performance, or maternal/pup behaviours during the lactation period in males and females of the parental or first generation. Caesarean section of selected 2000 ppm (both sexes treated) dams at GD 20 showed reduced foetal body weight and skeletal variations. Maternal exposure to toluene at 2000 ppm caused decreased pup weights throughout lactation in F1 and F2 animals.

Overall the NOAEC for parental toxicity and off-spring toxicity was 500 ppm (1875 mg/m3). The NOAEC for effects on fertility was 2000 ppm (7500 mg/m3), the highest dose tested.