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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
not conducted under GLP, published in peer-reviewed literature, fully adequate for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Principles of method if other than guideline:
Principle of test:
- to determine if benzyl derivatives (including mixed xylene and the m-, o- and p-xylene isomers) can cross the placenta, reaching the foetal blood and amniotic fluid in concentrations proportional to that in the maternal blood or in the atmosphere.
- to evaluate if the tested organic solvents have embryotoxic effects.
- to investigate if the incidence of minor or major abnormalities increase following exposure to these organic solvents.

Short description of test conditions:
- CFY Rats - groups of rats were were exposed to inhalation of xylene at 250, 1900 or 2400 mg/m^3. Tthe animals were killed by ether anaesthesia on gestation day 21. Samples of maternal and foetal blood and amniotic fluid were collected for sample determination via a Hewlett-Packard gas chromatograph.
- CFLP Mice - groups of mice were exposed to mixed xylene at 500 and 1000 mg/m^3 or, ortho-, meta-, and para-xylene at 500 mg/m^3 atmospheric concentrations for 24h/day continuously or for four hours three times a day intermittently from day 6-15 of gestation. On the 18th day of pregnancy, the animals were killed by ether anaesthesia.
- NZW Rabbits - exposed to mixed xylene, ortho-, meta-, para-xylene at 0, 500 or 1000mg/m^3 atmospheric concentrations for 24h/day from day 7-20 of gestation. On the 30th day of pregnancy, the animals were killed by ether anaesthesia.

Parameters analysed / observed:
- Rats - maternal and foetal blood and amniotic fluid were collected for testing for the presence of the tested substances. Foetuses were examined for developmental effects and the mothers were observed for post-implantation loss.
- Mice - foetuses were observed for developmental abnormalities.
- Rabbits - maternal toxicity and foetal development was examined
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
60.2% m-xylene, 13.6% p-xylene, 9.1% o-xylene, and 17% ethylbenzene
Xylenes (mixed) was obtained from the Shell Oil Company (Houston, Texas) in a single lot #.F-3091
Specific details on test material used for the study:
The composition of the mixed xylene and the purity of the xylene isomers used in the study were not specified.

Test animals

Species:
other: Rats, mice, rabbits
Strain:
other: CFY rats, CFLP mice, New Zealand white rabbits
Details on test animals and environmental conditions:
RATS
- Source: Lati-Gödöllo, Hungary
- Strain: Female CFY

MICE
- Source: LATI-Gödöllo, Hungary
- Strain: Female CLFP

RABBITS
- Source: BUKISZ, Budapest, Hungary
- Strain: Female New Zealand white rabbits
- Weight at study initiation: 3-4 kg
- The day of mating was considered to be the first day of gestation

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
not specified
Vehicle:
unchanged (no vehicle)
Details on exposure:
Details were not provided in the publication

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): not specified
- Storage temperature of food: not specified

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:not specified
- Method of holding animals in test chamber:not specified
- Source and rate of air:not specified
- Method of conditioning air:not specified
- System of generating particulates/aerosols:not specified
- Temperature, humidity, pressure in air chamber: not specified
- Air flow rate: not specified
- Air change rate: not specified
- Method of particle size determination: not specified
- Treatment of exhaust air:not specified

TEST ATMOSPHERE
- Brief description of analytical method used: not specified
- Samples taken from breathing zone: not specified

Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Rats were mated in a harem system and the first day of gestation was taken as they day of finding sperm in the vaginal smear.
Mice were mated in a harem system and the first day of gestation was finding a vaginal plug.
Rabbits mated and the day of mating was considered to be day 1 of pregnancy.
Duration of treatment / exposure:
RATS:
- day 7-15 of gestation

MICE:
- day 6-15 of gestation

RABBITS:
- day 7-20 of gestation
Frequency of treatment:
RATS
- Gestation day 7-15 for 24h/day - mixed xylene. The rats were killed by ether anaesthesia on the 21st day of pregnancy.

MICE
- Gestation day 6 -15 - 4 hours 3 times a day - ortho-xylene, meta-xylene, para-xylene, mixed xylene
- On the 18th day of pregnancy, the animals were killed by ether anaesthesia.

RABBITS
- Gestation day 7-20 for 24h/day. The animals were killed by ether anaesthesia on the 30th day of pregnancy.
Duration of test:
RATS:
- day 7-21 of gestation - the animals were exposed to test substances on gestation days 7-15 and sacrificed on gestation day 21

MICE
- day 7-18 gestation - the animals were exposed from day 7-15 of gestation and then sacrified on the 18th day of gestation

RABBITS
- day 7- 30 gestation - the animals were exposed to test substances on gestation days 7-20 and then sacrificed on day 30
Doses / concentrationsopen allclose all
Remarks:
Mixed xylene (rats, 24h/day, day 7-15 gestation)
Dose / conc.:
500 mg/m³ air (analytical)
Remarks:
Ortho-xylene (mice, 4 hours 3 times/day, day 6-15 of gestation)
Dose / conc.:
500 mg/m³ air (analytical)
Remarks:
Meta-xylene (mice, 4 hours 3 times/day, day 6-15 of gestation)
Dose / conc.:
500 mg/m³ air (analytical)
Remarks:
Para-xylene (mice, 4 hours 3 times/day, day 6-15 of gestation)
Remarks:
Mixed xylene (mice, 4 hours 3 times/day, day 6-15 of gestation)
Remarks:
Ortho-xylene (rabbits, 24h/day, day 7-20 of gestation)
Remarks:
Meta-xylene (rabbits, 24h/day, day 7-20 of gestation)
Remarks:
Para-xylene (rabbits, 24h/day, day 7-20 of gestation)
Remarks:
Mixed xylene (rabbits, 24h/day, day 7-20 of gestation)
No. of animals per sex per dose:
Number of dams in each groups:
RATS
- mixed xylene: 250mg/m^3 = 23, 1900 mg/m^3 = 22, 3400 mg/m^3 = 19
- Controls (air) = 20

MICE
- ortho-xylene: 500 mg/m^3 = 17
- meta-xylene: 500 mg/m^3 = 18
- para-xylene: 500 mg/m^3 = 17
- mixed xylene: 500mg/m^3 = 15, 1000 mg/m^3 = 15
- Controls (air) = 115

RABBITS
- ortho-xylene: 500 mg/m^3 = 9
- meta-xylene: 500 mg/m^3 = 9
- para-xylene: 500 mg/m^3 = 10, 1000 mg/m^3 = 8
- xylene: 500 mg/m^3 = 10, 1000 mg/m^3 = 10
- Controls (air) = 60
Control animals:
yes, concurrent no treatment

Examinations

Maternal examinations:
DETECTION OF THE FIRST DAY OF GESTATION
- Rats - checking for sperm in the vaginal smear
- Mice - checking for a vaginal plug

BODY WEIGHT: Yes

POST-MORTEM EXAMINATIONS: Yes
- presence of test substances in maternal blood and related signs of toxicity
- checking for dose-dependent toxic effects
Ovaries and uterine content:
Examinations included:
- Presence of post-implantation loss: Yes
- Presence of abortions: Yes
Fetal examinations:
- External examinations: Yes (body weight/weight retardation)
- Soft tissue examinations: Yes (anomalies of uropoetic apparatus)
- Skeletal examinations: Yes (including skeletal retardation, presence of extra bones)
- Presence of test substances in foetal blood and amniotic fluid: Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rats: maternal toxic effects of mixed xylene were moderate and dose-dependent.
Mice: no toxicity seen at exposure to substances at 500 mg/m^3 concentration
Rabbits: maternal toxicity was not apparent at 500 mg/m^3
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Rats: 1 died when exposed to 3400 mg/m^3 mixed xylene for 24h/day day 7-15 of gestation
Mice: no mortality was observed for mice exposed to mixed xylene or xylene isomers
Rabbits:1 for para-xylene 1000 mg/m^3 and 3 for mixed xylene 1000 mg/m^3
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Rabbits: mild toxic effects - mixed xylene and each of the xylene isomers tested at 1000 mg/m^3 decreased maternal weight gain
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
Rabbits: mixed xylene and each of the xylene isomers tested at 1000 mg/m^3 concentration caused a loss in the number of foetuses by abortion
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Rats: mixed xylene increased post-implantation loss.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
% dead or resorbed significant results (p < 0.05) were seen in:
- Rats : mixed xylene at 3400 mg/m^3 (13%)
- Please refer to the more detailed table attached (Table 1)
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
% dead or resorbed significant results (p < 0.05) were seen in:
- Rats : mixed xylene at 3400 mg/m^3 (13%)
- Please refer to the more detailed table attached
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
dose level: Mxed xylene and xylene isomers 24h/day
Remarks:
Rabbits
Effect level:
ca. 1 000 mg/m³ air (analytical)
Basis for effect level:
number of abortions
Remarks on result:
other: Caused spontaneous abortions
Dose descriptor:
dose level: Mixed xylene 24/day
Remarks:
Rats
Effect level:
ca. 3 400 mg/m³ air (analytical)
Basis for effect level:
mortality
Remarks on result:
other: 1 died
Dose descriptor:
dose level: Para-xylene, mixed xylene
Remarks:
Rabbits
Effect level:
ca. 1 000 mg/m³ air (analytical)
Basis for effect level:
mortality
Remarks on result:
other: 1 for para-xylene, 3 for mixed xylene

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Rats: at the highest concentrations of mixed xylene, there was a decrease in the body weight in male foetuses.
Mice: exposure to mixed xylene and each of the xylene isomers caused an increase in the incidence of weight retarded foetuses at least at the higher concentration.
Rabbits: mixed xylene and xylene isomers at 1000 mg/m^3 concentration often caused a decrease in the weight of female foetuses and exposure at 500 mg/m^3 caused a moderate embryotoxic effect where there was an increased incidence of weight retardation.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
not examined
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Rats: mixed xylene caused foetal skeletal retardation at each concentration, particularly increased at the higher concentrations. The highest concentrations of mixed xylene increased the incidence of extra ribs.
Mice: exposure to mixed xylene and xylene isomers caused skeletal retardation.
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Mixed xylene and xylene isomers did not prove to be teratogenic under the doses tested in rats, mice and rabbits in this study.

Effect levels (fetuses)

open allclose all
Dose descriptor:
dose level: Mixed xylene and xylene isomers 24h/day
Remarks:
Rabbits
Effect level:
ca. 1 000 mg/m³ air (analytical)
Sex:
female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: Decrease in the weight of foetuses
Dose descriptor:
dose level: Mixed xylene 24h/day
Remarks:
Rats
Effect level:
>= 3 400 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: Decrease in male foetal body weight
Remarks:
13% (p <0.05)

Fetal abnormalities

open allclose all
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
The highest concentrations of mixed xylene increased the incidence of extra ribs in mice foetuses
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal retardation and malformation
Description (incidence and severity):
Rats: mixed xylene caused foetal skeletal retardation at each concentration tested, particularly increased at the higher concentrations.
Mice: exposure to mixed xylene and each of the xylene isomers caused skeletal retardation.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

The study identified 500 mg/m^3 as a NOAEL for effects on foetal survival and foetal malformations or variation for rabbits.

Applicant's summary and conclusion

Conclusions:
Based on the results of the experiment in accordance with a method similar to the OECD Guideline 414 (Prenatal Developmental Toxicity Study) Guideline, it can be concluded that mixed xylene and the o-, p- and m-xylene isomers produced no teratogenic effects in mice, rats or rabbits. Increased post-implantation loss in rats at higher concentrations was observed and in rabbits exposed to 1000 mg/m^3 xylene and the o-, p- and m-xylene isomers caused abortion. There was also a significant case of maternal toxicity: one rat died when exposed to 3400 mg/m3, four rabbit dams died - one for para-xylene 1000 mg/m3 and three for mixed xylene 1000 mg/m3, three aborted, four showed total resorptions, and a decrease in maternal weight gain was observed. Abortion in rabbits at 1000 mg/m3 was also seen with all other solvents looked at in this study.
Executive summary:

The embryotoxic effects of mixed xylene and the o- , p- and m- xylene isomers were investigated in mice, rats and rabbits following a method similar to the OECD Guideline 414 (Prenatal Developmental Toxicity Study) Guideline.

 

Groups of CFY rats were exposed to the inhalation of mixed xylene at 250, 1900 or 3400 mg/m^3 for 24hday from day 7 - 15 of gestation. CFLP mice and NZ rabbbits were exposed to inhalation of 500 mg/m^3 ortho, meta- , para- xylene and 500 or 1000 mg/m^3 mixed xylene for 24h/day from gestation days 6 - 15 . Untreated animals served as controls, which inhaled pure air.

 

Mixed xylene and all the xylene isomers crossed the placenta and were found present in foetal blood and amniotic fluid. Maternal toxic effects at all solvent concentrations were moderate and dose- dependent. Mixed xylene increased post- implantation loss in rats. Mortality was observed in rats at 3400mg/m^3 mixed xylene and at 100mg/m^3 para- xylene and xylene in rabbits. No mortality was observed in mice exposed to mixed xylene or any of the xylene isomers. Mixed xylene and xylene isomers at 1000 mg/m^3 decreased maternal weight gain in rabbits and increased the number of foetal losses by abortion. A significant percentage of dead or resorbed foetus were seen in rats at 3400 mg/m^3 mixed xylene.

 

In terms of foetal effects, the highest concentrations of mixed xylene (≥3400 mg/m^3 = 13% (p<0.05) in rats caused a decrease in the body weight in male foetuses. Mixed xylene and all xylene isomers increased the incidence of weight retarded foetuses in mice, especially at the higher concentrations. When mixed xylene and xylene isomers were tested at 1000 mg/m^3 in rabbits, they caused a decrease in the weight of female foetuses and exposure at 500 mg/m^3 caused a moderate embryotoxic effect where there was an increased incidence of weight retardation.