Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status not known, non-guideline animal experimental study, limitations in design and/or reporting but otherwise adequate for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
One generation reproduction study in CD rats.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Crl-CD® (SC) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., St. Constance, Ontario, Canada
- Age at study initiation: 50 days
- Weight at study initiation: group mean range: 223-226 g (males), 162-165 g (females)
- Fasting period before study: none
- Housing: During the study, animals were caged individually in stainless steel wire mesh cages, with the exception of mating (nightly co-housing of males with females) and during lactation (female with the litter during non-exposure intervals)
- Diet: Purina Laboratory Chow 5002 (mash) ad libitum except during exposure periods
- Water: tap water ad libitum
- Acclimation period: 20 days (June 3-23, 1981)
- On day 21 of gestation, each female's cage was fitted with a stainless steel floor pan; these were removed on day 14 of lactation.
- Litter Kleen® hardwood shavings were added to the females cages on day 21 of gestation and fresh bedding provided as necessary. Bedding was removed on day 14 of lactation.

ENVIRONMENTAL CONDITIONS
- Temperature: 63-80°F (>90% of values between 70-75%)
- Humidity: 13-68% (>85% of values between 30-68%)
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 24 June 1981 To: 4 January 1982
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
The test material was measured out using a graduated cylinder into an Ehrlenmeyer flask. The test material was pumped to a JSS spraying systems atomiser using an FMI lab pump (model RPG-20) with a piston. Delivery lines used were teflon tubing. The test material was atomised with compressed air at a back pressure of 20 p.s.i and directed into the chamber inlet portal.
No further details reported.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2 or 1:1
- Length of cohabitation: Mating units (1:2) did not change for the first 8 days of the mating period. After this time, males were reassigned randomly among unmated females (by group), on 1:1 ratio. The total mating period was 21 consecutive days.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

A special mating was performed to provide F0 study males at 100 days of age, with mating experience prior to initiation of the actual study mating. However, all males regardless of reproductive performance were incorporated into the mating design for the main study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Miran determinations were confirmed by gas chromatography analysis.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
For 131 days prior to mating, with exposure continued in females on gestation days 1–20 and lactation days 5–20.
Details on study schedule:
- One-half of all F0 males were sacrificed after the mating period for gross postmortem examination; the remaining half were sacrificed and examined 21 days later.
- One-half of the group I F0 females and group IV F0 females were sacrificed on GD 21 for developmental toxicity evaluation.
- The remaining F0 females were allowed to deliver litters.
Remarks:
Doses / Concentrations:
0, 60, 250 or 500 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 60±2, 250±5 or 500±13 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
0 ppm - 30 males and 60 females, 60 ppm - 10 males and 20 females, 250 ppm - 10 males and 20 females. There were three high dose groups (500 ppm): group IV contained 20 males and 40 females and both sexes were treated; group V contained 10 males and 20 females but only males were treated; group VI contained 10 males and 20 females but only females were treated.
Control animals:
yes, sham-exposed
Details on study design:
Twenty control and twelve 500 ppm females were killed on day 21 of gestation and foetuses were evaluated for external, soft tissue and/or skeletal malformations. The remaining females from all groups were allowed to deliver their litters and offspring were evaluated for growth and survival during a 21 day lactation period.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, pre- and post-exposure

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the pre-mating period. Males and unmated females were weighed weekly throughout the mating and post-mating periods.
- Mated females weighed on days 1, 7, 13, 19 and 21 of gestation.
- Females with litters weighed on days 1, 4, 14 and 21 of lactation.

FOOD CONSUMPTION: Yes
- recorded at 2-day intervals during gestation for all mated females.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 post partum: yes. Litters were standardized by pooling all pups within each treatment group on lactation day 4 and redistributing four males and four females from this pool to each dam. However, on some days the pups could not be pooled if only one litter was available. In this case, litters were culled to four males and four females when possible.

PARAMETERS EXAMINED
The following parameters were examined offspring:
- Pups were weighed, sexed, and given a gross external examination on lactation days 1, 4, and 21.
- Randomly selected pups from each group (one/sex/litter) and all remaining F0 females with litters were sacrificed on day 21 of lactation and subjected to gross necropsy.
- The remaining pups were maintained for the postweaning interval of 28–49 days and weighed and sacrificed on day 49.
- Randomly selected pups from each group (one/sex/litter) were given a complete gross postmortem examination.

GROSS EXAMINATION OF DEAD PUPS:
- yes, gross external examination and the stomach was evaluated for the presence of milk. Visceral contents of thoracic and abdominal cavities examined.
- Pups found dead prior to day 4 of lactation were stored in 70% ethanol. pups found dead on day 5 or later were examined and discarded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: One-half of all F0 males were killed after the mating period for gross post mortem examination; the remaining half were killed and examined 21 days later.
- Maternal animals: One-half of the group I F0 females and group IV F0 females were killed on GD 21 for developmental toxicity evaluation. The remaining F0 females were killed on day 21 of lactation and subjected to gross necropsy.

POST-MORTEM EXAMINATIONS: Yes
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Half of the males in each group were killed after completion of the mating period. Testes, epididymides, seminal vesicles and prostate were stored in 10% neutral buffered formalin. The testes were weighed.
- The remaining males were killed 21 days later (December 14 1981). Abnormal tissue, testes, epididymides, seminal vesicles and prostate were stored in 10% neutral buffered formalin.

ORGAN WEIGHTS: The testes were weighed.
Postmortem examinations (offspring):
SACRIFICE
- Pups maintained for the postweaning interval of 28–49 days were weighed and killed on day 49. Randomly selected pups from each group (one/sex/litter) were given a complete gross post mortem examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- The following tissues were taken and stored in 10% neutral buffered formalin: adrenals, bone marrow, brain, eyes, gonads, epididymides, heart, colon, duodenum, ileum, kidneys, liver, lung, lymph node, mammary gland, pancreas, salivary gland, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, thyroid, urinary bladder, uterus, prostate, gross lesions, tissue masses and thymus.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Testes and ovaries from all pups subjected to gross examination on lactation day 21 and day 49 post-partum were weighed.
Statistics:
Appropriate evaluations were performed between data for the control and treated groups.
Reproductive indices:
Mating, fertility and pregnancy indices.
Offspring viability indices:
Pup survival and litter survival.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- A total of 12 females (1 group II, 3 group III, 4 group IV, 1 group V and 3 group VI) did not mate during the study. Evaluation of vaginal smears indicated that with the exception of 2 group IV females, all other females were not showing normal oestrus cycling during the mating period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- The female mating index in group III and group VI was significantly lower than for controls (85 and 85%, respectively, vs. 100% for controls), however, a similar effect was not observed in group IV (500 ppm exposed males and females) and there was also an unusually high mating performance in the controls.
- The male mating index, pregnancy rate, and fertility index in exposed animals were comparable to control values.
Dose descriptor:
NOAEC
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: no systemic toxicity or effects on reproduction
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
BODY WEIGHT (OFFSPRING)
- No statistically significant decrease in mean pup body weights were observed in the exposed versus control groups at days 1 and 14. On lactation day 4, mean pup weights were statistically significantly decreased in groups II (60 ppm), III (250 ppm), and IV (500 ppm) (post-pooling) when compared with controls, but the decreases (about 8%) were not of a biologically significant magnitude. The decreased weights may have been the consequence of an elevated mean pup weight in the control group potentially caused by a smaller mean litter size (mean number of live pups per litter: 9.6, 11.8, 12.5, 12.4, 10.8, and 11.8 for groups I–VI, respectively).
- Pups from group IV had statistically significant decreased mean pup weights on lactation day 21 (90% of controls) and statistically significant decreased terminal body weights at 49 days of age (as a percentage of controls: males, 92%; females, 93%). However, despite the marginal decreases observed in mean pup weights in group IV, no decreases in body weights were observed in pups from group VI, in which dams were exposed to the same concentration of xylene (500 ppm) for the same period of time as were dams in group IV. The marginal decreases observed in mean pup weights from group IV were considered not to be an adverse effect of treatment.

ORGAN WEIGHTS (OFFSPRING)
- Female pups from the mid- and high-dose groups (groups III and IV) also had statistically significant decreased absolute (76 and 78% of controls, respectively) and relative (80% and 84% of controls, respectively) ovary weights at 21 days of age, but the decreases were not concentration related and were not observed at 49 days of age. In addition, decreases in ovary weights were also not observed in group VI pups.
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
500 ppm mixed xylene (administered for 6 hours per day for 131 days prior to mating, during mating and continuing through gestation and lactation) is a NOAEC for systemic and reproductive toxicity.
Executive summary:

Groups of male and female CD rats were exposed to 0, 60, 250, or 500 ppm mixed xylenes (groups I, II, III, and IV, respectively by inhalation. exposure was for 6 hours per day, 5 days per week, for 131 days prior to and during mating, with exposure continued in females on gestation days 1–20 and lactation days 5–20. Two additional 500 ppm groups were similarly exposed, except that only the F0 males were exposed in group V, and only the F0 females were exposed in group VI. In-life parameters evaluated in adults included body weights, observations for mortality and clinical signs, detailed weekly physical examination, maternal body weights and maternal food consumption and food efficiency. One-half of all F0 males were sacrificed after the mating period for gross post mortem examination; the remaining half were sacrificed and examined following a 21 day treatment-free period.

Litters were standardized by pooling all pups within each treatment group on lactation day 4 and redistributing (where possible) four males and four females from this pool to each dam. Pups were weighed, sexed, and given a gross external examination on lactation days 1, 4, and 21. Randomly selected pups from each group (one/sex/litter) and all remaining F0 females with litters were sacrificed on day 21 of lactation and subjected to gross necropsy. The remaining pups were maintained for the postweaning interval of 28–49 days and weighed and sacrificed on day 49. Randomly selected pups from each group (one/sex/litter) were given a complete gross postmortem examination.

The highest exposure level of 500 ppm mixed xylene administered for 6 hours per day for 131 days prior to mating, during mating and continuing through gestation and lactation is a NOAEC.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 171 mg/m³
Species:
rat
Quality of whole database:
Studies conducted in rats demonstrate no evidence that mixed xylene or ethyl benzene adversely affect reproduction.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Mixed xylene (CAS 1330-20-7) comprises individual xylene isomers (m-xylene, o-xylene, p-xylene) and <10% ethyl benzene.

Non-human information

In a one-generation reproductive toxicity study (Bio/dynamics Inc., 1983), groups of male and female CD rats were exposed to 0, 60, 250, or 500 ppm technical-grade xylene (comprising 2.4% toluene, 12.8% ethyl benzene, 20.3% p-xylene, 44.2% m-xylene, 20.4% o-xylene) by inhalation for 6 hours per day, 5 days per week, for 131 days prior to mating, with exposure continued in females on gestation days (GDs) 1–20 and lactation days 5–20. The highest exposure level of 500 ppm mixed xylene, administered for 6 hours per day for 131 days prior to mating, during mating and continuing through gestation and lactation, is a NOAEC for all endpoints measured. There was no evidence from this study that mixed xylene containing 12.8% ethyl benzene is a reproductive toxicant.

Additional information is available on ethyl benzene. Similarities in the physicochemical and toxicological properties of ethyl benzene and xylenes, together with toxicokinetic information indicating that biotransformation proceeds primarily via hydroxylation of the alkyl side chain followed by urinary excretion of conjugated metabolites, suggests that read-across between these structures is scientifically justifiable. (See section 13 of the IUCLID dossier for further details.)

In a 2-generation study conducted on ethyl benzene (Faber et al., 2006), male and female Crl:CD(SD)IGS BR rats were exposed by inhalation to 0, 25, 100 or 500 ppm ethyl benzene vapour (for 6 hr/day) for at least 70 consecutive days prior to mating, with exposure of the females continuing through gestation up to GD20 after which ethyl benzene was administered via oral gavage (26, 90 or 342 mg/kg/day in corn oil) on lactation days 1 and 4. The gavage treatment was designed to provide a similar maternal blood concentration (AUC) to that achieved by inhalation. At 500 ppm ethyl benzene, the males of both generations showed reduced body weight gain and increased liver and kidney weights whilst in high dose females only liver weights were affected. As the reduced body weight gain in the 500 ppm males was transient, and since there were no histopathological changes associated with the increased organ weights found in the 500 ppm males and females, these changes were considered not to be adverse. Therefore, for parental systemic toxicity the NOEC was considered to be 100 ppm and the NOAEC 500 ppm. There were no adverse effects of ethyl benzene on reproductive performance in either generation, nor were spermatogenic endpoints, ovarian follicle counts, reproductive organ weights or macroscopic pathology affected. The NOAEC for reproductive toxicity and offspring development was 500 ppm ethyl benzene.

Overall these studies provide no evidence that mixed xylene or ethyl benzene have a potential to adversely affect reproduction in the rat. A NOAEC greater than 500 ppm (greater than 2171 mg/m3) was obtained in both instances.

Human information

No relevant information


Short description of key information:
Available animal data provide no evidence of an adverse effect on sexual function, fertility or development.

Justification for selection of Effect on fertility via inhalation route:
No adverse effects on sexual function, fertility or development were apparent in a rat one generation study conducted on mixed xylene (NOAEC exceeds 2171 mg/m3; Bio-Dynamics, 1983). A two generation study on ethyl benzene was also negative (NOAEC 2171 mg/m3; Faber et al., 2000).

Effects on developmental toxicity

Description of key information
Information is available on the effect of mixed xylene on prenatal development in rats exposed to concentrations up to and including 2000 ppm (8684 mg/m3). The results indicate that maternal toxicity (reduced corrected maternal body weight gain) occurred at exposures that were lower than those causing a biologically meaningful (>10%) reduction in foetal body weight, indicating that mixed xylene is not selectively toxic towards the foetus. 
Link to relevant study records
Reference
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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
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)
- 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:
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: a constant rate of liquid chemical was delivered (with a HPLC pump for 2000 ppm or with an infusion pump for the other concentrations). This liquid chemical was delivered at the top of a heated glass column filled with glass beads. Compressed air heated by a glass heater was introduced at the bottom of the glass column in a counter-current fashion to the liquid flow. 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 80°C. Concentrations of technical xylene were obtained by adding the analytical concentrations of ethylbenzene, o, m- and p-xylene.
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.
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 - killed 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.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality: No maternal deaths.
Bodyweight: At 1000 ppm, non-significant (33%) reduction in corrected body weight gain on GD6-21; at 2000 ppm, significant (97%) reduction in corrected body weight gain on GD6-21.
Food consumption: At 2000 ppm, significant reduction throughout exposure.
Dose descriptor:
BMCL10
Remarks:
mixed xylenes
Effect level:
887 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
BMCL10
Remarks:
mean, all xylene isomers
Effect level:
780 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No effects on mean number of implantation sites and of live foetuses, and the incidence of non-live implants and resorptions.
Foetal body weights showed a dose-related decrease (significantly different from the control at 500 ppm and above). The decrease was approximately 16% at 2000ppm, 7% at 1000 ppm and 4% at 500 ppm.
The occurrences of foetuses with external, visceral and skeletal variations did not differ between the control and the treatment groups. Visceral malformations occurred sporadically in one or two foetuses and were distributed across the different groups.
Dose descriptor:
BMCL10
Remarks:
Mixed xylenes
Effect level:
1 082 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Dose descriptor:
BMCL10
Remarks:
mean, all xylene isomers
Effect level:
1 094 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The BMC10 for maternal toxicity was 887 ppm, and the BMC10 for foetal effects was 1082 ppm. 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 from gestation days 6-20 to technical xylene resulted in maternal toxicity at 2000 ppm. There was a 4% lower foetal body weight at 500 ppm, which is considered to be of limited biological relevance. Technical mixed xylenes were not teratogenic up to 2000 ppm.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
BMCL10
4 698 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Studies conducted in rats provide no evidence that mixed xylene is selectively toxic to the foetus.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Mixed xylene (CAS 1330-20-7) comprises individual xylene isomers (m-xylene, o-xylene, p-xylene) and <10% ethyl benzene.

A well-documented guideline study (Saillenfait et al, 2003) evaluated the developmental toxicity of mixed xylene (comprising 15.3% ethylbenzene, 21.3% o-xylene, 43.9% m-xylene, 19.4% p-xylene); m-, o- and p-xylene isomers; and ethyl benzene in Sprague Dawley rats following whole-body exposure to nominal vapour concentrations of 0, 100, 500, 1000, or 2000 ppm for 6 hours/day from gestation days (GD) 6-20. For rats exposed to mixed xylene, there was a dose-dependent decrease in maternal corrected body weight gain (body weight gain during GD 6-21 minus gravid uterine weights), including a 33% decrease in corrected body weight gain at 1000 ppm, although statistical significance was achieved only at 2000 ppm (-97% reduction compared to control rats). Maternal food consumption during GD 6-21 were also reduced at 1000 (-8% compared to controls) and 2000 ppm (-16% compared to controls), although statistical significance was only seen at the highest dose group. Lower foetal body weights were observed in all treatment groups, with statistically significant reductions observed at higher exposures (-4% at 500 ppm; -7% at 1000 ppm; -16% at 2000 ppm). However it is noted that the magnitude of some of these changes was small (<10%), suggesting that their biological relevance is limited. There was no indication of any teratogenic effect in this study and no indication of foetal dysmorphogenesis. Dose-response data for foetal body weights and corrected maternal weight gain from this study were subsequently analyzed by the Registrant using Benchmark Dose (BMD) modelling software developed by US-EPA (2013). The intention was to better characterize exposure levels associated with biologically relevant reductions in foetal weight and maternal corrected body weight gain. The data were analyzed using a range of models included in the software package, and tested for goodness-of-fit using a chi-square goodness-of-fit test (Chi-square p-value < 0.10 indicated inadequate fit). The best-fit model was selected, and the BMCL10 (reflecting the 90% lower confidence limit on the BMD) calculated. The underling calculations are included in the IUCLID dataset. The results showed that the BMCL10 for maternal toxicity (assessed as a decrease in corrected body weight gain) was 887 ppm for mixed xylene (equivalent to 3851 mg/m3) and in a range 616 - 898 ppm for the individual xylene isomers; the BMCL10 for foetal effects (assessed as a decrease in foetal weight) was 1082 ppm for mixed xylene (equivalent to 4698 mg/m3) and 965 – 1306 ppm for the individual isomers. Hence maternal toxicity occurred at exposures that were lower than those causing a biologically meaningful (>10%) reduction in foetal body weight indicating that mixed xylene (and other xylene isomers) are not selectively toxic towards the foetus.

The prenatal developmental toxicity of mixed xylene was also assessed by Hass and Jakobsen (1993). In this study pregnant female rats were exposed whole-body to nominal atmospheric vapour concentrations of 0 or 200 ppm for 6 hours/day from gestation days 4-20. No adverse effects were apparent in the dams, hence 200 ppm was the NOAEC for maternal toxicity. Delayed ossification of the os maxillare was significantly increased in litters from treated dams and was judged by the study authors to be treatment-induced. However, since the definitive assessment of Saillenfait clearly demonstrates the absence of this finding following exposure to higher (and lower) concentrations of both mixed xylene and xylene isomers it is therefore considered to be incidental to treatment.

With regard to any potential effect of in utero exposure to mixed xylene on foetal body weight, Hass and Jakobsen (1993) found no difference in weight at birth for female pups, while male pup body weights were slightly (+8%; statistically significant) increased in litters from the 200 ppm treatment group. Since this level of exposure is intermediate between the low- and no effect level for foetal body weight effects reported by Saillenfait et al, 2003), it indicates that the NOAEC for developmental toxicity of mixed xylene is at least 200 ppm / 868 mg/m3.

The postnatal developmental toxicity of mixed xylene was assessed in a series of three studies by Hass et al, (1993, 1995, 1997) using pregnant Wistar rats exposed to a single concentration of xylene by inhalation.

In the first study (Hass and Jakobsen, 1993) pregnant female rats were exposed whole-body to nominal atmospheric vapour concentrations of 0 or 200 ppm for 6 hours/day from gestation days 4-20. As noted above, 200 ppm was a NOAEC for maternal toxicity. Some deterioration in Rotorod test performance was reported for treated females on PND 22, 23 and 24 while the results for treated males were indistinguishable from those of the controls. However when discussing these findings the study authors note that “the animals were not tested blindly to exposure group and not on the same day. Therefore, a possible influence from the experimenter cannot be totally excluded”, hence no reliable LOAEC or NOAEC can be derived from the study.

In the second study (Hass et al., 1995), pregnant female rats were exposed whole-body to nominal atmospheric vapour concentrations of 0 or 500 ppm for 6 hours/day from gestation days 7-20. No maternal effects were apparent at 500 ppm, which is therefore a NOAEC for maternal toxicity. Assessment of post-natal development of the pups included reflex development, neurobehavioural/neuromotor ability and learning/memory. One male and one female from each litter were kept in pairs of the same sex in standardized housing from 22 days of age until 3 months, when they underwent the Morris water maze test. Another male and female from each litter were kept in enriched housing, 4-5 per sex per cage (cages contained various toys) and tested for rotarod (the ability to remain on a rotating rod for 30 seconds), open field, and Morris maze performance at about 3 months of age. A non-statistically significant decrease in rotarod performance was reported in exposed female pups. Offspring from xylene-exposed rats that were raised in the enriched environment showed no difference in the Morris maze test when compared with controls but offspring from exposed rats that were raised in the standard housing had impaired performance. At 16 weeks, exposed offspring took more time to find a platform hidden in the centre of the pool; the effect was limited to the female offspring from the standard housing. These females had an increase in swimming length, but swim speed was unaffected.

In the third study (Hass et al., 1997) pregnant female rats were exposed whole-body to nominal atmospheric vapour concentrations of 0 or 500 ppm for 6 hours/day from gestation days 7-20. 500 ppm was a NOAEC for maternal toxicity. Post-natal learning and memory abilities (Morris water maze test) were assessed in female offspring in standard housing conditions at 28 and 55 weeks of age. At 28 weeks, an increased latency for finding a platform that was moved to a new position was observed only during the first trial of a three-trial testing block, whereas the next two trials resulted in similar latencies between exposed and control rats. The increased latency again corresponded with increased swimming length. There were no differences at 55 weeks.

The results of the second and third studies suggested that prenatal exposure to 500 ppm xylenes, 6 hours per day on GDs 7–20 affected the performance of standard housing female rats in the Morris water maze test; a longer time was taken to find a hidden platform as swim length (i.e. the distance covered before finding the platform) was increased and swim speed was unaffected. The data suggest this is not a motor effect but a minimal effect on neurological development which was reversible. There were limitations in the design and/or reporting of these studies, including the use of only one concentration of mixed xylene in each. In addition, effects were only seen in one sex, were mitigated by differences in housing conditions and did not reflect an inability of animals to learn or recall the task as all animals reached the platform. Consequently this minor, reversible effect on performance is considered to be insufficiently robust to be used to determine a LOAEC.

A recent and well conducted 2-generation study on ethyl benzene included an evaluation of several developmental landmarks in the F1 and F2 offspring and an assessment of developmental neurotoxicity in the F2 offspring (Faber et al., 2006; 2007). Similarities in the physicochemical and toxicological properties of ethyl benzene and xylenes, together with toxicokinetic information indicating that biotransformation proceeds primarily via hydroxylation of the alkyl side chain followed by urinary excretion of conjugated metabolites, suggests that read-across between these structures is scientifically justifiable. (See section 13 of the IUCLID dossier for further details.)

In this investigation, concentrations of 0, 25, 100 or 500 ppm ethyl benzene were administered by inhalation for 6 hours /day throughout the study (except on lactation days 1-4 inclusive when the equivalent dose was administered to the parent females by oral gavage). The following physical developmental landmarks: pinna detachment, hair growth, incisor eruption and eye opening were assessed prior to weaning and vaginal patency and balano-preputial separation were assessed post weaning. There was no adverse effect of ethyl benzene on the acquisition of these landmarks in either the F1 or F2 generations. For the F2 generation, neurobehavioral development of one pup/sex/litter was also evaluated using the functional observational battery (on postnatal days 4, 11, 22, 45, and 60), motor activity (on postnatal days 13, 17, 21, and 61), acoustic startle testing (on postnatal days 20 and 60), Biel water maze learning and memory task (initiated on postnatal days 26 or 62). Whole-brain measurements, brain morphometric and histologic assessments were also conducted on postnatal days 21 and 72. No treatment-related effects were observed. The results of this 2 generation study, corroborate the results of the developmental toxicity study of Saillenfait et al. (2003), which also included exposure concentrations of 100 and 500 ppm ethyl benzene, in that there was no effect on the number of live foetuses or mean foetal weight and similarly no effect on the number of live pups per litter or mean pup body weight. Although ethyl benzene was not evaluated by Hass and co-workers (1993; 1995; 1997), the results from the 2-generation study provide good evidence in support of the lack of effect of mixed xylenes on neurobehavioural development or on brain development. Overall it is concluded that mixed xylene and ethyl benzene are not developmental toxicants.


Justification for selection of Effect on developmental toxicity: via inhalation route:
Information is available on the effect of mixed xylene on prenatal development in rats exposed to concentrations up to and including 2000 ppm (8684 mg/m3; Saillenfait et al., 2003). Analysis of dose-response data for foetal body weights and corrected maternal weight gain using Benchmark Dose (BMD) modelling software developed by US-EPA showed that the BMCL10 for foetal effects (assessed as a decrease in foetal weight) was 1082 ppm (4698 mg/m3), and the BMCL10 for maternal toxicity (assessed as a decrease in corrected body weight gain) was 887 ppm (3851 mg/m3). Hence maternal toxicity occurred at exposures that were lower than those causing a biologically meaningful (>10%) reduction in foetal body weight, indicating that mixed xylene and xylene isomers are not selectively toxic towards the foetus.

Justification for classification or non-classification

Mixed xylenes, xylene isomers and ethylbenzene do not warrant classification for reproductive or developmental toxicity according to DPD or CLP.

There are sufficient data available on component substances to conclude that streams within this class are not reproductive or developmental toxicants and do not require a label for these endpoints.