Reaction mass of ethylbenzene and m-xylene

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 status unknown, guideline study, published in peer reviewed literature, no restrictions, fully adequate for assessment.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2003

Reference 2

Reference Type:

publication

Title:

Benchmark Dose Technical Guidance Document

Author:

US-EPA

Year:

2010

Bibliographic source:

Available from: http://www.epa.gov/raf/publications/pdfs/benchmark_dose_guidance.pdf

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

o-xylene

EC Number:

202-422-2

EC Name:

o-xylene

Cas Number:

95-47-6

Molecular formula:

C8H10

IUPAC Name:

o-xylene

Details on test material:

≥ 99.5% pure

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l'Arbresle, France)
- Body weight at study initiation: 180 - 200 g
- Housing: Individually 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 exposures
- Water: filtered tap water ad libitum except during exposures
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21±2°C
- Humidity: 50±5%
- Photoperiod: 12 hrs dark / 12 hrs light:

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 with dynamic and adjustable laminar air flow (6-8 m3/h), maintained at a negative pressure of ≤3 mm water.
- System of generation: The system consisted of passing an additional air-flow rate through the fritted disk of a heated bubbler containing the test chemical. Chamber concentrations were obtained by changing the temperature of the bubbler and/or the air flow rate through the fritted disk of the bubbler. The vaporized compounds were introduced into the main air-inlet pipe of the exposure chambers.
- Temperature, humidity, pressure in air chamber: 23±2°C, 50±5%

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography with a flame ionization detector
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentrations were determined by gas chromatography with a flame ionization detector. The column temperature was maintained at 100°C. The concentrations determined by analyses were essentially the same as the target concentrations.

Details on mating procedure:

Nulliparous females were housed overnight with adult males (one male to two or three females) from the same strain and supplier. The day that vaginal smears were found to be sperm-positive was considered day 0 of gestation (GD).

Duration of treatment / exposure:

6 hr/day

Frequency of treatment:

Daily, from day 6 through 20 of gestation.

Duration of test:

21 days

No. of animals per sex per dose:

23 - 26 mated females/group; 20 - 26 pregnant females/group

Control animals:

yes, concurrent vehicle

Details on study design:

Control animals were exposed concurrently to filtered room air in an adjacent chamber identical to those of the treatment groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes - recorded on GD 0, 6, 13 and 21
- Body weight changes were calculated for the following gestation intervals: 0-6, 6-13 and 13-21.
- The corrected weight gain was the body weight gain between GD 6-21 subtracted from gravid uterus weight.

FOOD CONSUMPTION: Yes
- Measured for the intervals GD 6-13 and 13-21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Uterus

Ovaries and uterine content:

The uterus was removed and weighed. The number of corpora lutea, implantation sites, resorptions, and dead and live foetuses were recorded. Uteri with no visible implantation sites were stained with ammonium sulphide (10%) to detect very early resorptions.

Fetal examinations:

Live foetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live foetuses from each litter were preserved in Bouin's solution and examined for internal soft tissue changes. The other half were fixed in ethanol (70%), eviscerated, and then processed for skeletal staining with Alizarin Red S for subsequent skeletal examination.

Statistics:

Where appropriate, the data were presented as mean ± SD. One-way analysis of variance was used to analyse the number of corpora lutea, implantation sites and live foetuses, maternal food consumption and body weights and was followed by Dunnett's test where differences were found. The Kruskal-Wallis test was used to evaluate the percentages of non-live implants, resorptions, and males, and the proportions of foetuses with alterations in each litter and was followed by the Mann-Whitney test where appropriate. Pregnancy rates and percentages of litters with any malformations or with external, visceral or skeletal variations were analysed using Fisher's test. Least-squares analysis was carried out where applicable. The level of statistical significance reported was P<0.05. The litter was the unit of analysis for 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:
Mortalities: None
Clinical signs: Hypotonia and somnolence at 2000 ppm
Bodyweight: Bodyweight decreased on GD 21 at 1000 ppm and on GD 13 and GD 21 at 2000 ppm. Significant decreases in maternal weight gain during the whole exposure period and in corrected weight gain at 1000 and 2000 ppm. Corrected weight gain significantly decreased at 1000 and 2000 ppm (by 3.8 and 11.5% respectively).
Food consumption: Significantly reduced during the first half of exposure at 1000 ppm and throughout the exposure period at 2000 ppm.

Effect levels (maternal animals)

Dose descriptor:

BMCL10

Remarks:

o-xylene

Effect level:

720 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Numbers of implantations, live foetuses, non-live implants, resorptions, foetal sex: no effects
Foetal bodyweights: a concentration-related reduction (significantly different from control at 500 ppm (by 5%) and at higher concentrations).
External, visceral and skeletal effects: Single cases of visceral malformations at 500 and 1000 ppm-exposed groups. Several common external, visceral and skeletal variations in the control and test groups. The occurrence of foetuses (total number and mean per litter) with skeletal variations (all types) was significantly elevated above control at 2000 ppm.

Effect levels (fetuses)

Dose descriptor:

BMCL10

Remarks:

o-xylene

Effect level:

965 ppm (nominal)

Based on:

test mat.

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

Intergroup comparison of the incidence of skeletal variations

(Table based on Saillenfait AM et al, 2003, Food and Chemical Toxicology 41 415-429,Table 6)

	Exposure level (ppm)
	0	100	500	1000	2000
skeletal exam Total no foetuses (litters)	144 (21)	142 (21)	169 (24)	133 (20)	134 (19)
No (%) foetuses with skeletal variations	26 (18.1)	23 (16.2)	40 (23.7)	37 (27.8)	47 (35.1) ##
Mean % foetuses with skeletal variations per litter	18.2 ±18.6	18.3 ±20.5	23.9 ±22.2	27.3 ±16.7	36.7 ±24.4 **
## Significant difference from control (air), P < 0.01 Fisher's test. ** Denotes significant difference from control (air), P < 0.01, Mann-Whitney test.

Applicant's summary and conclusion

Conclusions:

The BMC10 for maternal toxicity was 720 ppm (3.1 mg/L), and the BMC10 for foetal effects was 965 ppm (4.2 mg/L). Hence maternal toxicity (as indicated by a reduction in corrected maternal body weight gain) occured at exposures that were lower than those resulting in a biologically meaningful (>10%) reduction in foetal body weight.

Executive summary:

Inhalation exposure of Sprague-Dawley rats to 0, 100, 500, 1000 or 2000 ppm o-xylene from gestation days 6 -20 resulted in maternal toxicity at 1000 (4.4 mg/L) and 2000 ppm (8.8 mg/L). Foetal toxicity at 1000 and 2000 ppm, in the presence of maternal toxic effects was seen. There was a 5% difference in foetal weight at 500 ppm (2.2 mg/L), which is considered to be of limited biological relevance, and o-xylene was not teratogenic up to 2000 ppm.