Distillates (petroleum), cracked stripped steam-cracked petroleum distillates, C8-10 fraction

Developmental toxicity / teratogenicity

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, guideline animal experimental study, published in peer reviewed literature, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)

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:

- Source: BDH Ltd, Poole, Dorset, UK
- Purity: 99.9%
- Physical state: clear colourless liquid

Test animals

Species:

rat

Strain:

other: Crl: CD (SD) BR VAF/Plus

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9-10 wk
- Weight at study initiation: 170-229 g
- Diet: ad libitum except during exposure
- Water: ad libitum except during exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 20±2°C
- Humidity: 60 ± 20%
- Air changes (per hr): not reported
- Photoperiod: 12hrs dark / 12hrs 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
Toluene vapour generated by metering toluene from a central pressurised reservoir onto a sintered glass disc contained within a glass vessel through which dried, filtered air was flowed. The resulting vapour was introduced into the inlet duct of the exposure chamber.
At the end of 6 h exposure, chambers were cleared with clean air for 20 min, after which test animals were removed to their holding cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TEST ATMOSPHERE
Chamber atmospheres analysed approximately hourly during exposure and analysed by GC-FID. Nominal concentration calculated daily, by dividing weight of test material used by total airflow in chambers during exposure.
Target and achieved chamber concentrations were similar throughout the exposure period; analysed concentrations were within 1-4% of nominal.
Homogeneity of chamber atmospheres was verified during pre-exposure trials by sampling from seven different locations within each chamber.

Details on mating procedure:

- Mating: time mated to males (14-15 wk old) of the same strain
- Proof of pregnancy: sperm in vaginal smear or presence of a vaginal plug

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

gestation day 6-15 (GD 6-15) where day of positive smear/vaginal plug was taken as day gestation day 0 (GD0)

Duration of test:

20 days

No. of animals per sex per dose:

25 females per group

Control animals:

yes, sham-exposed

Details on study design:

RATIONALE FOR DOSE SELECTION
12 presumed-pregnant females/group exposed (whole body) to 0, 500, 1000, 2000, 3500 or 5000 ppm toluene for 6 h/day on GD 6-15 as part of a range-finding study. Dose-related maternal and developmental toxicity at exposures of ≥2000 ppm, including decreased maternal and foetal body weight and post-implantation loss.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Dams observed twice daily before and after exposure for clinical signs, changes in general appearance and mortality.
During exposure, 8 different animals were observed each day at half-hour intervals in the inhalation chamber. Cage positions were rotated daily to assure uniform exposure of all rats. Any altered behavioural signs and response to tapping on the chamber wall (to monitor alertness) were recorded.

BODY WEIGHT:
Presumed pregnant rats were weighed on GD0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. Final GD20 bodyweights were corrected for gravid uterine weight.

FOOD CONSUMPTION:
Food consumption was measured from weigh day to weigh day, beginning on GD0.

WATER CONSUMPTION:
Water consumption was monitored daily.

POST-MORTEM EXAMINATIONS:
Killed on GD20 - gross abnormalities assessed.

Ovaries and uterine content:

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Uteri from females that appeared non-gravid and individual uterine horns without visible implantations were opened and placed in 10% ammonium sulphide solution for visualisation of implantation sites.

Fetal examinations:

- External examinations: all per litter
- Soft tissue examinations: half per litter
- Skeletal examinations: half per litter

Statistics:

Statistical methods included: ANOVA followed by intergroup comparisons using Williams' or Shirley’s tests - body weight, adjusted maternal body weight, maternal body weight changes, water and food consumption; Kruskal-Wallis test with Shirley's test or t-tests - mean number of corpora lutea, implantations, pre- and post-implantation loss, early and total foetal death, live foetuses, litter weight, mean foetal weight, gravid uterus weight, sex ratio, and most sternebral variants; mixed model ANOVA using litter as the basis for analysis- foetal body weights.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
3000 ppm: Ataxia, hunched posture, uncoordinated gait, limb tremor, closed eyelids, hyper-responsivity, increased water intake, decreased food consumption and reduced maternal body weight gain from initiation of exposure to GD20.
1500 ppm: Ataxia, hunched posture, uncoordinated gait, closed eyelids, hyper-responsivity and reduced maternal body w eight gain during the exposure period only (GD6-15).

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEC

Effect level:

750 ppm (nominal)

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEC

Effect level:

2 812 mg/m³ air (nominal)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Gravid uterus weight, mean litter weight and mean foetal body weights were statistically significantly lower than controls at 3000 ppm and there was a higher incidence of unossified or reduced sternebra. Mean foetal bodyweights were statistically significantly decreased at 1500 ppm and a slightly increased incidence of sternebral variants (unossified, reduced).

No malformations occurred in the control or 750 ppm group animals, however, the incidence of total events in the 250, 1500 and 3000 ppm groups was increased significantly. Soft tissue anomalies occurred most frequently in controls. Skeletal anomalies occurred at similar incidences in all groups other than 750 ppm. The most frequent observation involved reduced and/or no ossification of sternebrae (primarily 5 and 6) in foetuses from groups 250, 1500 and 3000 ppm (statistically significant, for 1500 and 3000 ppm groups).

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEC

Effect level:

750 ppm (nominal)

Basis for effect level:

other: Developmental Toxicity

2

Key result

Dose descriptor:

NOAEC

Effect level:

2 812 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

Adjusted maternal bodyweight (ie excluding uterine contents) on GD20:

(Table based on Roberts LG et al, 2007, Reproductive Toxicology 23, 521-531,Table 3)

	0 (control) (n=24)	250 ppm (n=22)	750 ppm (n=20)	1500 ppm (n=19)	3000 ppm (n=22)
Mean adjusted maternal bodyweight (g)	270	273	271	274	249**

Mean maternal body weight change (g):

(Table based on Roberts LG et al, 2007, Reproductive Toxicology 23, 521-531,Table 3)

Gestation day	0 (control) (n=24)	250 ppm (n=22)	750 ppm (n=20)	1500 ppm (n=19)	3000 ppm (n=22)
0 - 6	31.3	31.8	33.8	36.1	34.1
6 - 10	18.8	19.9	16.5	12.3**	- 4.5**
6 - 16	55.1	56.9	52.0	47.4*	20.6**
16 - 20	50.4	50.9	49.4	53.6	53.9
6 - 20	105.5	107.8	100.4	101.3	74.4**

* P 0.05;  ** P0.01

Incidence of malformations and anomalies

There were no increases in the incidence of specific or total malformations with increased exposure and low incidences (<2.5%) of malformations in toluene exposed groups were considered to be incidental to treatment.

Summary of reproduction and mean foetal weight data:

There were no adverse effects on implantations, number and viability of foetuses or foetal sex distribution.

Table based on Roberts LG et al, 2007, Reproductive Toxicology 23, 521-531,Table 4

	0 (control)	250 ppm	750 ppm	1500 ppm	3000 ppm
No. females mated	25	25	25	25	25
No. pregnant (%)	24 (96)	22 (88)	20 (80)	19 (76)	23 (92)
No. with viable foetuses	24	22	20	19	22
Corpora lutea	14.0	14.4	14.6	14.9	14.8
Implantation sites	12.0	13.3	12.2	13.4	13.6
Gravid uterus weight (g)	66.7	64.7	61.5	62.5	58.6
Pre-implantation loss index % (mean)	8.3	7.5	16.6	9.5	8.1
No. viable foetuses	293	272	233	236	277
Foetal deaths (mean)	0.6	0.9	0.5	1.0	1.0
Post-implantation loss % (mean)	5.3	7.0	4.7	7.6	7.2
Mean litter size	12.2	12.4	11.7	12.4	12.6
No. litters with resorptions %	9	11	7	10	13
Sex distribution (% males)	47.6	50.5	45.2	45.3	48.4
Mean litter weight (g)	41.4	41.0	39.8	39.7	37.9*
Mean body weight of viable foetuses	3.47	3.32*	3.44	3.20**	3.02**
male	3.59	3.41*	3.50	3.29**	3.10**
female	3.39	3.23	3.38	3.13**	2.93**
Pre-implantation loss = (corpora lutea - implants) / (corpora lutea)  Post-implantation lost = (implants - live foetuses) / (implants) * P 0.05; ** P0.01

Foetal sternebral variants

There were slightly increased incidences of unossified sternebra at 3000 and 1500ppm.

Table based on Roberts LG et al, 2007, Reproductive Toxicology 23, 521-531,Table 7

	Toluene concentration (ppm)
	0	250	750	1500	3000
No. of litters	24	22	20	19	22
Foetuses examined	146	131	116	116	138
Sternebral variants - Unossified (No./Mean %)	55/37	57/44	48/37	65/53	83/60*
Sternebral variants - Reduced (No./Mean %)	37/29	60/45	36/31	49/44	61/45*
Sternebral variants - Total with variants (No./Mean %)	77/55	94/72	69/57	89/75*	106/77

* P 0.05

Applicant's summary and conclusion

Conclusions:

The maternal NOAEC for toluene in pregnant Sprague Dawley rats, exposed whole-body during gestational days 6-15 (inclusive), 6h/day, was 750 ppm (2812 mg/m3). The foetal NOAEC was 750 ppm (2812 mg/m3).

Executive summary:

The developmental toxicity of toluene was evaluated following whole body inhalation (6 h/day) in pregnant Sprague Dawley rats exposed to toluene (99.9% pure) at concentrations of 0, 250, 750, 1500 or 3000 ppm from GD6-15. Doses were selected based on a preliminary study performed over a range of concentrations from 0 to 5000 ppm in which maternal and foetal toxicity was observed at 2000 ppm and above.

Toluene induced clinical signs in pregnant dams included ataxia, hyper-responsivity, increased water intake, decreased food consumption and lower body weight gain at 3000 ppm. At 1500 ppm ataxia, hyper-responsivity and reduced body weight gain during the exposure period were seen. 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. Low incidences (2.5%) of various malformations occurred in the 250, 1500 and 3000 ppm groups, however, there was no exposure-related increase in the incidence of specific or total malformations and thus these findings were not attributed to toluene.

Based on these observations the NOAEC for maternal toxicity and for foetal toxicity was 750 ppm (2812 mg/m3).