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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
days 0-21 of gestation
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP status unknown, guideline animal experimental study, published in peer reviewed literature, no restrictions, fully adequate for assessment
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
not specified
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
Molecular formula:
Constituent 2
Reference substance name:
Details on test material:
99% pure obtained from Fluka Chemie AG (Bucks, Switzerland)

Test animals

Details on test animals or test system and environmental conditions:
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l'Arbresle, France)
- Details: nulliparous females
- Weight at study initiation: 180-200 g
- Fasting period before study: none
- Housing: Mated females were housed singly in clear polycarbonate cages with stainless-steel wire lids and corn cob granules as bedding.
- Diet: Food pellets (UAR Alimentation Villemoisson, France) ad libitum except during exposure
- Water: filtered tap water ad libitum except during exposure
- Acclimation period: 2 weeks

- Temperature: 21 ± 2°C
- Humidity: 50 ± 5%
- 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
unchanged (no vehicle)
Details on exposure:
Exposures were conducted in 200 liter glass/stainless steel inhalation chambers with dynamic and adjustable laminar air flow (5-10 m3/h). The chambers were maintained at a negative pressure of no more than 3 mm water. The chamber temperature was 23 ± 0.6 °C and the relative humidity 57 ± 5%. Chemical vapours were generated by passing an additional airflow rate through the fritted disk of a heated bubbler containing the test chemical. Under these conditions the vapourized compound were carried out into the main air inlet pipe of the exposure chambers. The concentrations were adjusted by varying the airflow passing through the fritted of the bubbler.

- Brief description of analytical method used: Actual concentrations were measured once daily by collecting atmosphere samples through glass tubes packed with activated charcoal. Pseudocumene was desorbed with carbon disulfide and analysed by a gas chromatograph using ethylbenzene as internal standard. Samples were chromatographed on a column full of PEG 20 M and 2m length, which was maintained at a temperature of 130 °C. Another gas chromatograph was used for the continuous monitoring of the levels of exposure; this latter chromatograph was equipped with a flame ionization detector and an automatic gas-sampling valve. Concentration measurements were performed at regular intervals (0.5 min).
As pseudocumene has a low vapour pressure (1.83 mm Hg at 22 °C) the presence of liquid particles was evaluated at the highest concentration generated (i.e. 900 ppm). Airborne particles were measured with an optical counter of particles with a lower detection limit of 0.75 µm.

- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The actual concentrations (±SD) (from charcoal tubes) were 100 (±5), 299 (±11), 592 (±17), and 896 (±19) ppm pseudocumene.
Intra-day variations were <4.3%.
No difference in particles counts was observed between the clean filtered air (control) and vapour-laden air in the exposure chamber
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male : 2-3 females
- Length of cohabitation: overnight until evidence of mating
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
15 days
Frequency of treatment:
6 h/day, on days 6 through 20 of gestation.
Duration of test:
Following acclimatisation and mating, the study lasted from day 0-21 of gestation.
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 100, 300, 600, and 900 ppm
nominal conc.
Doses / Concentrations:
0, 492, 1470, 2950 and 4430 mg/m3
nominal conc.
based on 1 ppm being approx 4.92 mg/m3 (Korsak et al, 1997)
Doses / Concentrations:
100 ±5, 299 ±11, 592 ±17, and 896 ±19 ppm
analytical conc.
No. of animals per sex per dose:
24-25 bred female rats (17-24 pregnant)
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on results from:
(i) McKee et al, 1990. 3-generation reproduction study , rats exposed to vapours of a C9 aromatic hydrocarbon mixture containing significant proportions of trimethylbenzenes (corresponding to c.a. 41.7, 200, and 600 ppm pseudocumene). No effects on male and female fertility. Offspring growth affected at the mid and high exposure concentrations. Mice were also exposed to same C9 fraction (i.e. 40.5, 202, or 613 ppm pseudocumene) and at the high concentration there was maternal toxicity and evidence of developmental toxicity.

(ii) Ungvary et al, 1983. A decrease in the body weight of male fetuses and a fetal ossification delay were reported following inhalation exposure of pregnant rats to a C9 aromatic hydrocarbon mixture (containing approximately 70-140 ppm) from day 7 to 15 of gestation.

(iii) Lehotzky et al, 1985. In a behavioural developmental toxicity study, rats were exposed to 600, 1000, or 2000 mg/m3 aromatol (a mixture of methyl-ethyl benzenes and trimethylbenzenes). No significant effects were observed in dams and offsprings.


Maternal examinations:
- Time schedule: Not reported
- Time schedule for examinations: gestation days 0, 6, 13 and 21
- Time schedule: gestation day periods 6-13 and 13-21
- Sacrifice on gestation day 21
- Organs examined: Uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
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
- Foetal weight: Yes
Fetal examinations:
- External examinations: Yes: all per litter (including the oral cavity)
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Whenever possible, the data were presented as mean ± SD. The number of corpora lutea, implantation sites and live fetuses and various body weights were analyzed by one-way analysis of variance, followed by Dunnett's test if differences were found. The frequency of post-implantation loss, dead fetuses, resorptions and alterations among litters was evaluated by using the Kruskal-Wallis test followed by the Mann-Whitney test where appropriate. Rates of pregnancy and incidences of fetal alterations per dose were analysed by using Fisher's test. Where applicable, least-squares analysis was carried out. The reported level of statistical significance was p < 0.05. The litter was used as the basis for the analysis of fetal variables.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All dams survived to their scheduled euthanization. No clinical signs attributed to pseudocumene were noted in any of the treatment groups. Maternal weight gain was significantly reduced during the first half of exposure at 600 ppm, and throughout the exposure period at 900 ppm (4430 mg/m3). Significant decrease in corrected weight gain was observed at 600 and 900 ppm. Food consumption was significantly depressed during the whole exposure period at 600 and 900 ppm (2950 and 4430 mg/m3). There were no significant changes in the average numbers of implantations and live fetuses, in the incidence of post-implantation loss and resorptions, or in fetal sex ratio across groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
1 470 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
2 950 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
1 470 mg/m³ air (nominal)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
Effect level:
2 950 mg/m³ air (nominal)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Fetal body weights were significantly lower than control at 600 and 900 ppm (5% and 11-12%, respectively). Fetal examination revealed single cases of malformations in the 300, 600 and 900 ppm groups (i.e. diaphragmatic hernia or missing ribs). External variations (club foot) were only seen in the control group. There was no significant effect of treatment on the incidence of visceral or skeletal variations.

Effect levels (fetuses)

Key result
Dose descriptor:
Effect level:
4 430 mg/m³ air (nominal)
Basis for effect level:
other: teratogenicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

The NOAEC for maternal and developmental toxicity was 300 ppm (1470 mg/m3).
Executive summary:

Pregnant rats were exposed whole body to vapours of pseudocumene (0, 100, 300, 600, and 900 ppm; 0, 492, 1470, 2950 and 4430 mg/m3), 6 h/day, on gestational days 6 through 20. Significant decrease in maternal body weight gain and food consumption was observed at concentrations of 600 ppm pseudocumene, or greater. Foetal toxicity, expressed as significant reduction in fetal body weight, occurred at 600 and 900 ppm pseudocumene. There was no evidence of embryolethal or teratogenic effects following inhalation exposure. In summary, the NOAEC for maternal and developmental toxicity was 300 ppm for pseudocumene.