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Toxicological information

Developmental toxicity / teratogenicity

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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 status unknown, guideline animal experimental study, published in peer reviewed literature, no restrictions, fully adequate for assessment
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2005

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
99% pure obtained from Fluka Chemie AG (Bucks, Switzerland)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- 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
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 200 L glass/stainless steel inhalation chambers
- Chambers maintained at negative pressure of no more than 3 mm water. Concentrations adjusted by varying airflow passing through the fritted of the bubbler.
- System of generation: vapours generated by passing an additional airflow rate through the fritted disk of a heated bubbler containing the test chemical. The vaporised compound was carried out into the main air inlet pipe of the exposure chambers.
- Chamber temperature and relative humidity: 23 ± 0.6 °C and 57 ± 5%.
- Air flow rate: Dynamic/adjustable laminar air flow (5-10 m3/h).

TEST ATMOSPHERE
- Brief description of analytical method used: Actual concentrations 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).
- The presence of liquid particles was evaluated at the highest concentration generated (i.e 900 ppm), as pseudocumene has a low vapour pressure (1.83 mm Hg at 22 °C).
- 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:
yes
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
Remarks:
Doses / Concentrations:
0, 100, 300, 600, and 900 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 492, 1470, 2950 and 4430 mg/m3
Basis:
other: target concentration (based on 1 ppm being approx 4.92 mg/m3 (Korsak et al, 1997)
Remarks:
Doses / Concentrations:
100 ±5, 299 ±11, 592 ±17, and 896 ±19 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
24-25 bred female rats (17-24 pregnant)
Control animals:
other: yes, air only
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 foetuses and a foetal 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 or offspring.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Not reported

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 13 and 21

FOOD CONSUMPTION: Yes
- Time schedule: gestation day periods 6-13 and 13-21

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- 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
Statistics:
Number of corpora lutea, implantation sites, live foetuses, body weights: one-way analysis of variance, followed by Dunnett's test if differences were found.
Post-implantation loss, dead foetuses, resorptions, alterations among litters: Kruskal-Wallis test followed by the Mann-Whitney test where appropriate.
Pregnancy rate, incidences of foetal alterations: Fisher's test, where applicable, least-squares analysis was carried out. The litter was used as the basis for the analysis of foetal variables.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Mortality: None
- Clinical observations: No treatment-related effects
- Bodyweight: 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: Significantly decreased during the whole exposure period at 600 and 900 ppm (2950 and 4430 mg/m3).
- No treatment-related effects on average numbers of implantations and live foetuses, the incidence of post-implantation loss and resorptions or foetal sex ratio.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
1 470 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEC
Effect level:
2 950 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
1 470 mg/m³ air (nominal)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEC
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:
- Foetal weight: Significantly lower than control at 600 and 900 ppm (5% and 11-12%, respectively)
- Malformations:Single cases of malformations present 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)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
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 foetal 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.