reaction mass of ethylbenzene and m-xylene and p-xylene

Endpoint summary

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

Type of information:

experimental study

Adequacy of study:

supporting 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 or test system 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

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 at the highest dose tested

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.

Reproductive effects observed:

not specified

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 xylenes or ethylbenzene adversely affect reproduction.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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% ethylbenzene, 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 of reproductive toxicity in this study.

LOA acknowledges that the one generation reproduction study (BioDynamics, 1983) in the dossier does not include some of the parameters measured in an OECD 443 (extended one-generation reproductive toxicity study). OECD 443 study in rats with p-xylene will be performed and read-across to o-xylene and m-xylene. The read-across is based on similarity of response of xylene isomers in sub-chronic and developmental toxicity studies. Also, Toxcast analysis on xylene isomers shows very similar responses and this read-across approach helps to reduce excessive and wasteful use of animals.

Human information

No human data are available.

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 xylenes (NOAEC exceeds 2171 mg/m3; Bio-Dynamics, 1983).

Effects on developmental toxicity

Description of key information

Information is available on the effect of individual xylene isomers (m-, o-, and p-xylene; mixed xylenes) on prenatal developmental toxicity at 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 xylene isomers are not selectively toxic towards the foetus. 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting 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

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 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:

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

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 (nominal)

Based on:

test mat.

Basis for effect level:

other: Developmental Toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The BMC10 for maternal toxicity was 887 ppm (3.85 mg/L), and the BMC10 for foetal effects was 1082 ppm (4.7 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 from gestation days 6-20 to technical xylene resulted in maternal toxicity at 2000 ppm (8.7 mg/L). There was a 4% lower foetal body weight at 500 ppm (2.2 mg/L), which is considered to be of limited biological relevance. Technical mixed xylenes were not teratogenic up to 2000 ppm.

Reference 2

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

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 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:

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.

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.

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.

Dose descriptor:

BMCL10

Remarks:

o-xylene

Effect level:

720 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:
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.

Dose descriptor:

BMCL10

Remarks:

o-xylene

Effect level:

965 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: Developmental Toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rabbit

Quality of whole database:

Studies conducted in rabbits provide no evidence that xylene isomers (o-, p- and m-xylene) are selectively toxic to the foetus.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

BMCL10

4 190 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Studies conducted in rats provide no evidence that xylene isomers (including mixed xylenes) are selectively toxic to the foetus.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Key studies

A well-documented guideline study (Saillenfait et al, 2003) evaluated the developmental toxicity of mixed xylenes (comprising 15.3% ethylbenzene, 21.3% o-xylene, 43.9% m-xylene, 19.4% p-xylene); m-, o- and p-xylene isomers; and ethylbenzene 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. Results for mixed xylenes and the xylene isomers are included in this dossier. Dose-dependent decreases in maternal corrected body weight gain (body weight gain during GD 6-21 minus gravid uterine weights) and maternal food consumption during GD 6-21 were observed. These were accompanied by dose-dependent reductions in foetal body weight, however the magnitude of some of these changes was small (<10%) suggesting that their biological relevance was 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 for mixed xylenes. The results showed that the BMCL10 for maternal toxicity (assessed as a decrease in corrected body weight gain) was in an overall range of 616 - 898 ppm (2675 - 3899 mg/m3), and the BMCL10 for foetal effects (assessed as a decrease in foetal weight)was 965 - 1306 ppm (4190 - 5671 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 xylene isomers are not selectively toxic towards the foetus.

In the Ungváry and Tátrai 1985 paper, groups of 10 pregnant New Zealand rabbits were each exposed by inhalation to 0, 115, or 230 ppm mixed xylenes (0, 500, or 1000 mg/m3) or xylene isomers, for 24 hours per day on gestation days 7-20.  The composition of the mixed xylene used in the study was not specified. Mixed xylene and the o-, p- and m-xylene isomers produced no teratogenic effects in mice, rats or rabbits. The overall conclusion was that inhalation of 230 ppm mixed xylenes for 24 hours per day on gestation days 7–20 produced severe maternal toxicity in the NZW rabbit that impaired its’ ability to deliver live foetuses.  Maternal toxicity was not apparent at 115 ppm and therefore 115 ppm mixed xylene was considered to be the NOAEL for both maternal and developmental toxicity in the NZW rabbit.

In the Britton (2020), McGregor (2020), and Hackford (2020) OECD 414 studies, the three xylene isomers were tested in the rabbit using the same conditions and the same range of concentrations (0, 30, 100, 300 mg/kg bw/day). All xylene isomers elicited a similar maternal toxicity profile at the top dose with comparable transient post-dose observations being: change in ear colour and rapid breathing. M-xylene administration resulted in an initial body weight loss, but no effect on overall body weight gain or food intake. O-xylene administration resulted in lower body weight gain and food intake. P-xylene administration resulted in similar effects to maternal body weight gain and food intake as for o-xylene with the only difference being the lower foetal weights compared to the controls. Overall , it can be concluded based on the weight of evidence that there was no adverse effect on pregnancy, embryonic or foetal development at any dose level for either of the xylene isomers. Therefore, based on these findings, the Lowest-Observed-Effect-Level (LOEL) and No Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 300 mg/kg/day and the No-Observed-Adverse-Effect-Level (NOAEL) for embryo-foetal development was considered to be 300 mg/kg/day.

 

Supporting studies

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.

 

With regard to any potential effect of in utero exposure to mixed xylenes 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 xylenes is at least 200 ppm / 868 mg/m3. LOA has cited some concerns in this paper. Firstly, it is unclear what was the technical dose of xylene used (200 or 500 ppm) and it is unclear why water was removed during the exposure period (6h/day GD4 -20; whole-body inhalation chamber), since it is not a common procedure in such studies. At study day 3, the body weight of adults in both groups (placebo and 200 ppm) were low (178 and 176g, respectively), compared to that observed on day 0 (in the range of 180 -200g). The number of animals tested postnatally was considered to be low (12 animals per group), and one litter born 2 days later was omitted from testing. There seems to be inconsistencies with the number of litters tested in different parts of study between the material and methods section and what is stated in the tables. There was no abnormal sex ratio reported or discussed in the text, which is inconsistent with table 5, where an abnormal ratio is reported in the placebo group, but is normal in the controls and treated groups. The physical development of pups were slightly advanced in the treatment groups, whereas they were slightly retarded in the control group when compared to the norm for the postnatal age. For the rotarod test, animals were not tested in a blinded manner nor were they tested on the same day.

 

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, neurobehavioral/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.

There were also concerns raised in this paper by LOA including why food was removed in the exposure period and why exposure was started at GD7, whilst animals were exposed at GD4 in the Hass 1993 study. The paper reports a lower absolute brain weight at PND28, whereas the relative brain weight is the same for the control and 500 ppm groups and therefore the difference in brain weight is related to an effect on body weight. The effects on post-natal bodyweight are inconsistent with an earlier study conducted by the same group. The appearance of the air righting reflex was significantly delayed in the exposed litters on PND15. On PND16, 4 pups in 4 litters from the exposed group were not able to air right, despite being able to air right on PND15. They were re-tested on PND17 and scored positive. The air righting reflex appears during the second week of life on approximately PND8 and is not fully developed until at least PND18 (Developmental and Reproductive Toxicology: A Practical approach, Third Edition, Ed. Ronald D Hood, Informa Healthcare, CRC press, 2012). The surface righting reflex was not different between the study groups in an earlier study (Hass and Jakobsen, 1993). No significant rotarod effects were observed at 500 ppm when tested in a blinded manner. At PND24 (the only common testing timepoint in Hass and Jakobsen, 1993 and Hass et al., 1995), the control group animals showed a large difference in rotarod effect between Hass 1993 and Hass et al. 1995, indicating a large variation in rotarod endpoint in the author’s lab. The above clearly indicates that the rotarod effects reported by ECHA, based on Hass and Jakobsen (1993) and attributed to xylene exposure, are false. In the Morris water maze test, no xylene-related effects where seen in animals that had been housed in an enriched environment (ie, larger cages with various toys). On the non-enriched animal group there is an effect reported when the platform is moved to the center of the pool. Data is seen to be inconsistent when comparing length values (cm and swim speed (cm/sec) in Table 7 values (e.g. 258 cm / 21 cm/sec = 12 sec and this is not in the bar in the figure; similarly 200 cm / 16 cm/sec = 12.5 sec). From Table 2, 15 animals should have been in the treated group in Figure 4, whereas only 14 swim paths are shown. In the treatment group, the average swim time ranged from 4.1 to 13.4 seconds (Table 6) and average swim distance ranged from 72-258 cm. The average swim path data is largely affected one female animal in the treatment group who showed a swim time of 41 seconds and a distance of 945 cm.

 

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. The experimenter was "blind" to the animal's exposure group and exposed and control animals were tested alternately in groups of two to four. In the beginning of the learning part of the test (week 12), the xylene-exposed offspring are reported to have used slightly more time to find the platform (Figure 1, blocks 1 and 2, p = 0.059). Figure 1 in Hass et al. (1997) appears to be exactly the same as the figure 2 in Hass et al. (1995), as is the P value reported in these two publications. Therefore, LOA concludes this is the same data used twice, however the study is reported as if this was a new study. 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. LOA also questions how the blinding procedure could have been maintained since there were only two groups and they were tested alternately and therefore the experimenter might know that the next group for testing is different from the one just tested. There were no exposure-related differences in offspring body weights during the entire study period. This provides further supports to LOA’s interpretation that pub bodyweight (and hence absolute brain weight claims) reported in Hass et al. (1995) could not be reproduced under exactly the same test conditions. Moreover, effects observed in Hass 1995 (e.g. weight effects in pups, increased postimplantation loss in the control group) are reported in the Hass 1997 study to be not present. If this is one study then the data should be exactly the same on all endpoints assessed., but if they are 2 separate studies then it is unclear why the authors chose to obtain exactly the same Morris water maze test results, including the same p-value (while different data was obtained for the other parameters). LOA is of the opinion that there are critical concerns with the data in this study.

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. Overall it is concluded that xylene isomers are not developmental toxicants.

Justification for selection of Effect on developmental toxicity: via inhalation route: 

Information is available on the effect of individual xylene isomers (m-, o-, and p-xylene; mixed xylenes) on prenatal developmental toxicity at 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 in the range 965 – 1306 ppm for the different isomers, and the BMCL10 for maternal toxicity (assessed as a decrease in corrected body weight gain) was 616 – 898 ppm. Hence maternal toxicity occurred at exposures that were lower than those causing a biologically meaningful (>10%) reduction in foetal body weight, indicating that xylene isomers are not selectively toxic towards the foetus.

Justification for classification or non-classification

Xylene isomers (including mixed isomers) are considered not to warrant classification for reproductive or developmental toxicity according to CLP.