m-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:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

See supporting information in section 13

Reason / purpose for cross-reference:

read-across source

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 a needless and wasteless 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

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

Qualifier:

equivalent or similar to guideline

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 exposed to inhalation of xylene at 250, 1900 or 2400 mg/m^3. The 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

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.

Species:

other: Rats, mice, rabbits

Strain:

other: CFY rats, CFLP mice, New Zealand white rabbits

Details on test animals or test system 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

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

Dose / conc.:

250 other: mg/m^3

Remarks:

Mixed xylene (rats, 24h/day, day 7-15 gestation)

Dose / conc.:

3 400 other: mg/m^3

Remarks:

Mixed xylene (rats, 24h/day, day 7-15 gestation)

Dose / conc.:

1 900 other: mg/m^3

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)

Dose / conc.:

500 other: mg/m^3

Remarks:

Mixed xylene (mice, 4 hours 3 times/day, day 6-15 of gestation)

Dose / conc.:

1 000 other: mg/m^3

Remarks:

Mixed xylene (mice, 4 hours 3 times/day, day 6-15 of gestation)

Dose / conc.:

500 other: mg/m^3

Remarks:

Ortho-xylene (rabbits, 24h/day, day 7-20 of gestation)

Dose / conc.:

1 000 other: mg/m^3

Remarks:

Ortho-xylene (rabbits, 24h/day, day 7-20 of gestation)

Dose / conc.:

500 other: mg/m^3

Remarks:

Meta-xylene (rabbits, 24h/day, day 7-20 of gestation)

Dose / conc.:

1 000 other: mg/m^3

Remarks:

Meta-xylene (rabbits, 24h/day, day 7-20 of gestation)

Dose / conc.:

500 other: mg/m^3

Remarks:

Para-xylene (rabbits, 24h/day, day 7-20 of gestation)

Dose / conc.:

1 000 other: mg/m^3

Remarks:

Para-xylene (rabbits, 24h/day, day 7-20 of gestation)

Dose / conc.:

500 other: mg/m^3

Remarks:

Mixed xylene (rabbits, 24h/day, day 7-20 of gestation)

Dose / conc.:

1 000 other: mg/m^3

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

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

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/m3 concentration
Rabbits: maternal toxicity was not apparent at 500 mg/m3

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/m3 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

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Rabbits: mixed xylene and each of the xylene isomers tested at 1000 mg/m3 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/m3 (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/m3 (13%)
- Please refer to the more detailed table attached

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Key result

Dose descriptor:

dose level: Mixed 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

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.

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)

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.

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

The study identified 500 mg/m³ as a NOAEL for effects on foetal survival and foetal malformations or variation for rabbits.

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/m3 (1 mg/L) 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 (3.4 mg/L), 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/m³ for 24h day from day 7 - 15 of gestation. CFLP mice and NZ rabbbits were exposed to inhalation of 500 mg/m³ ortho-, meta- , para- xylene and 500 or 1000 mg/m³ 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 3400 mg/m³ (3.4 mg/L) mixed xylene and at 100 mg/m³ (0.1 mg/L) 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/m³ (1 mg/L) 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/m³ mixed xylene.

 

In terms of foetal effects, the highest concentrations of mixed xylene (≥3400 mg/m³ = 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/m³ in rabbits, they caused a decrease in the weight of female foetuses and exposure at 500 mg/m³ caused a moderate embryotoxic effect where there was an increased incidence of weight retardation.

 

Reference 3

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.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2019 to 20 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25th June 2018

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The test item, m-xylene, was received on 21 June 2019 and on arrival it was given the Sequani log reference number TI/2019/095. Details of the consignment received were:
Batch number STBH7130
Appearance Colourless liquid
Expiry date 05 September 2024
Quantity supplied 4 x 1L
Purity 99.8 %

The test item was stored at room temperature and a certificate of analysis for it is presented in the report

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Specification
Eighty-eight female New Zealand White rabbits were supplied by Envigo Global Services Inc., 310 Swampbridge Road, Denver, PA 17517, USA. Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation. The animals were approximately five months of age on arrival and on examination were found to be healthy. After at least three days acclimatisation, they were re-examined and confirmed to be suitable for use. On the first day of dosing the females weighed 2.48 to 3.93 kg.

Allocation and identification
Allocation to groups was performed using a stratified randomisation procedure based on individual body weights recorded on Day 0 of gestation at the suppliers (making sure that, where possible, females mated with the same male were spread across the groups). Each animal was uniquely identified by a subcutaneously implanted micro-identification device. To aid identification during procedures, the animals were ear marked with their own unique identification numbers using an indelible pen as necessary. This mark was placed on the opposite ear to that of the supplier's number.

Housing and environmental conditions
The animals were housed individually in perforated-floor cages suspended over paper-lined trays. The study room was illuminated to give a cycle of 12 hours light and 12 hours dark and was air-conditioned. The target ranges for temperature and humidity were 16 °C to 20 °C and 40 % to 70 %, respectively. Recorded values were within, or marginally outside, these limits.

Diet and water
A pelleted diet, Teklad 2030C supplied by Envigo RMS (UK) Limited and mains tap water were freely available. From the day of arrival, approximately 30 g of hay was provided each day to animals as enrichment. Hay was supplied by IPS Product Supplies Ltd., BCM IPS Ltd., London, WC1N 3XX, United Kingdom. Where considered necessary, when food intake was reduced, feeding regimens were adjusted to encourage and maintain gut motility; details of additional and moistened diet offered are retained within the raw data. Flavoured Bunny Blocks™ (supplied by LBS Servicing Biotechnology Ltd, PO Box 431, RH6 0UW, United Kingdom) were available and replaced each week.

Route of administration:

oral: gavage

Vehicle:

other: 1 % w/v methylcellulose (medium viscosity) with 1 % v/v Tween 80

Details on exposure:

The test item was formulated daily, for each group separately, as a suspension in 1 % w/v methylcellulose (medium viscosity) with 1 % v/v Tween 80. All formulations were handled within a chemical fume hood. A quantity of test item was weighed directly into a container, made up to final weight with vehicle and covered with parafilm as much as possible. Formulations were stirred and mixed with a laboratory homogeniser until a homogeneous suspension was achieved. On receipt in the animal unit, formulations containing the test item were briefly shaken to re disperse the test item and stirred for at least five minutes before the start of dosing and until the completion of their use for dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sets of samples (for analysis or for contingency) were taken from each test item formulation prepared throughout the study. Those dedicated for analysis used on the first day of dosing and at the end of the dosing period were sent to the Principal Investigator at Covance, for analysis (Analytical reference ST11FC-meta-xylene-02R, under Covance reference number: WV89CL) using validated method ST11FG. Reserve formulation samples were stored both refrigerated (2 to 8 °C) and frozen (-20 °C). All remaining samples were retained and discarded once the final formulation analysis results were accepted.

Details on mating procedure:

Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.

Duration of treatment / exposure:

OECD 414 Main study. From Day 6 to Day 27 of gestation, inclusive.

Frequency of treatment:

Once daily

Duration of test:

Day 6 of gestaion to Day 28 of gestation

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

A total of 88 animals were used in this study, the design of which is given in Table 1 (in any other info) below. Animals were dosed once daily from Day 6 to Day 27 of gestation, inclusive, by gavage, using a rubber catheter and disposable syringe at a constant dose volume of 5 mL/kg body weight. Individual doses were adjusted according to the most recent body weight.

Maternal examinations:

Clinical Observations
Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily. From the start of dosing, animals were observed before and shortly after dosing. On weekdays, animals were also observed at one hour after dosing and at the weekend they were observed either at one hour after dosing or at the end of the working day, whichever was sooner.

Body weight
Body weights were recorded on Day 0 of gestation by the supplier. At Sequani, body weights were recorded daily from Day 4 to Day 28 of gestation, inclusive.

Food intake
The amount of food consumed by each animal was recorded daily over Days 4 to 6 of gestation and every two days thereafter during gestation (see Appendix 13 for deviation).

Ovaries and uterine content:

The females were killed on Day 28 of gestation by an overdose injection of sodium pentobarbitone. The animals were weighed, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative.
The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained in 70 % IDA (industrial denatured alcohol) for approximately seven days and then transferred and retained in 10 % buffered formalin.
3.6.2. Uterine examination
For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses.
The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix.
The foetuses and their placentae were removed and the uterus and ovaries retained in 10 % buffered formalin.

Fetal examinations:

On Day 28 of gestation, live foetuses were killed by an intraperitoneal injection of sodium pentobarbitone solution, weighed and examined for external abnormalities. Approximately 50 % of the foetuses in each litter were decapitated and the heads fixed in Bouin's fluid for subsequent serial sectioning. The intact foetuses and the bodies of the decapitated foetuses were placed in 70 % IDA. Later in the day they were skinned, dissected, the viscera were examined and the sex recorded. They were eviscerated and then a coronal section was made through the head, along the frontal parietal suture and the brain examined. The carcasses were then cleared in potassium hydroxide, stained with Alizarin red S to visualise the ossified skeleton and examined. Structural congenital abnormalities that impair, or potentially impair, the survival or constitution of the foetus were classified as major abnormalities. Abnormalities that in isolation, do not impair the survival or constitution of the foetus but could potentially interfere with normal bodily function were classified as minor abnormalities. Alternative structures occurring regularly in the control population, which may be permanent or transient, were classified as variants. Foetuses with major external or visceral abnormalities were photographed. For archiving, all foetal heads fixed in Bouin's fluid were stored in 10 % buffered formalin and all skeletal specimens were stored in aqueous glycerol with thymol crystals (to prevent fungal growth).

Statistics:

Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1. Depending on the nature of the data set that was to be analysed, appropriate tests were applied, as indicated in the table below. Where parametric tests were appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied. See Table 2 in Any other info - methods for statistical tests applied. Proportions of foetuses affected were treated as continuous non-parametric data, using one-sided step-wise Jonckheere Tests. Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.

Indices:

Pre-implantation loss (%) = (no. of corpora lutea – no. of implantation sites) / no. of corpora lutea x 100
Post-implantation loss (%) = (no. of implantation sites – no. of live foetuses) / no. of implantation sites x 100

Mean pre- and post-implantation losses were calculated on a proportional litter basis. Mean foetal body weights were calculated separately by sex for each litter; group mean body weights were calculated (separately by sex) from the litter means. The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.

Historical control data:

Historical control data available see attached 'background material'.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Abnormally purple ears were observed in most females given 300 mg/kg/day throughout the dosing period, on Days 8 or 15 of gestation in four females given 100 mg/kg/day and in one female given 30 mg/kg/day on Day 15 of gestation. Rapid breathing was also seen in several females given 300 mg/kg/day throughout the dosing period, although less frequently than purple pinnae. These observations were seen immediately after dosing but were no longer present after one hour.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, there was a mean body weight loss between Days 6 and 9 of gestation; thereafter, body weight gain was similar to, or greater than Control, so that overall body weight gain was similar in all groups. There was no effect of m-xylene administration on terminal body weight adjusted for the weight of the gravid uterus.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of m-xylene administration on food intake.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

There were no deaths during the study. Abnormally purple ears were observed in most females given 300 mg/kg/day throughout the dosing period, on Days 8 or 15 of gestation in four females given 100 mg/kg/day and in one female given 30 mg/kg/day on Day 15 of gestation. Rapid breathing was also seen in several females given 300 mg/kg/day throughout the dosing period, although less frequently than purple pinnae. These observations were seen immediately after dosing but were no longer present after one hour. At necropsy, abnormal cream areas in the liver of Female 137 and dark areas in the lungs of Female 142 (both given 100 mg/kg/day) were observed. Abnormal red and/or cream areas of the stomach were seen in Females 149 and 152 also given 100 mg/kg/day and Females 126 and 129 given 30 mg/kg/day. There were no maternal necropsy findings at 300 mg/kg/day.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

With the exception of two females (one Control and one given 30 mg/kg/day), all females were pregnant. This resulted in 21, 21, 22 and 22 females with live foetuses on Day 28 of gestation in the Control group and groups given 30, 100 or 300 mg/kg/day, respectively.

Other effects:

not examined

Details on maternal toxic effects:

There were no deaths during the study. Abnormally purple ears were observed in most females given 300 mg/kg/day throughout the dosing period, on Days 8 or 15 of gestation in four females given 100 mg/kg/day and in one female given 30 mg/kg/day on Day 15 of gestation. Rapid breathing was also seen in several females given 300 mg/kg/day throughout the dosing period, although less frequently than purple pinnae. These observations were seen immediately after dosing but were no longer present after one hour. At necropsy, abnormal cream areas in the liver of Female 137 and dark areas in the lungs of Female 142 (both given 100 mg/kg/day) were observed. Abnormal red and/or cream areas of the stomach were seen in Females 149 and 152 also given 100 mg/kg/day and Females 126 and 129 given 30 mg/kg/day. There were no maternal necropsy findings at 300 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

Remarks on result:

other: no other effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no effect of m-xylene on foetal or placental weights

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No changes in number of live offspring

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No changes in sex ratio observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No changes in litter size or weight observed

Changes in postnatal survival:

not examined

Description (incidence and severity):

Foetuses were killed on Day 28 of gestation

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed

Key result

Developmental effects observed:

no

Treatment related:

no

Table 1 Body weights (kg) - Group Mean Values

Sex: Female		Body Weight (Day of Gestation)
Group		0		4		5		6 (#)		7
Group: 1 Control 0 mg/kg/day	Mean	3.072		3.038		3.083		3.113		3.126
	SD	0.200		0.204		0.198		0.218		0.225
	N	21		21		21		21		21
	Trend	.		.		.		↑		.
Group: 2 m-xylene 30 mg/kg/day	Mean	3.110		3.066		3.121		3.159		3.184
	SD	0.258		0.273		0.273		0.260		0.277
	N	21		21		21		21		21
Group: 3 m-xylene 100 mg/kg/day	Mean	3.154		3.156		3.193		3.218		3.226
	SD	0.244		0.254		0.273		0.273		0.282
	N	22		22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	3.092		3.100		3.148		3.168		3.167
	SD	0.261		0.268		0.282		0.291		0.286
	N	22		22		22		22		22
	Trend	.		.		.		> 0.05		.
Sex: Female		Body Weight (Day of Gestation)
Group		8		9		10		11		12
Group: 1 Control 0 mg/kg/day	Mean	3.128		3.140		3.159		3.178		3.204
	SD	0.225		0.211		0.207		0.206		0.203
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Group: 2 m-xylene 30 mg/kg/day	Mean	3.166		3.151		3.152		3.171		3.200
	SD	0.273		0.262		0.264		0.265		0.253
	N	21		21		21		21		21
Group: 3 m-xylene 100 mg/kg/day	Mean	3.230		3.236		3.247		3.258		3.285
	SD	0.292		0.279		0.288		0.268		0.264
	N	22		22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	3.152		3.153		3.171		3.174		3.195
	SD	0.281		0.284		0.268		0.252		0.239
	N	22		22		22		22		22
	Trend	.		.		.		.		.
Sex: Female		Body Weight (Day of Gestation)
Group		13		14		15		16		17
Group: 1 Control 0 mg/kg/day	Mean	3.237		3.274		3.299		3.311		3.332
	SD	0.203		0.201		0.195		0.192		0.190
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Group: 2 m-xylene 30 mg/kg/day	Mean	3.233		3.266		3.291		3.302		3.309
	SD	0.246		0.243		0.246		0.260		0.259
	N	21		21		21		21		21
Group: 3 m-xylene 100 mg/kg/day	Mean	3.310		3.340		3.373		3.392		3.394
	SD	0.264		0.260		0.270		0.275		0.265
	N	22		22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	3.231		3.263		3.288		3.310		3.314
	SD	0.236		0.236		0.235		0.234		0.232
	N	22		22		22		22		22
	Trend	.		.		.		.		.
Sex: Female		Body Weight (Day of Gestation)
Group		18		19		20		21		22
Group: 1 Control 0 mg/kg/day	Mean	3.345		3.359		3.380		3.397		3.416
	SD	0.183		0.180		0.192		0.191		0.197
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Group: 2 m-xylene 30 mg/kg/day	Mean	3.323		3.349		3.371		3.390		3.408
	SD	0.259		0.264		0.256		0.253		0.257
	N	21		21		21		21		21
Group: 3 m-xylene 100 mg/kg/day	Mean	3.413		3.419		3.450		3.471		3.499
	SD	0.271		0.269		0.268		0.261		0.268
	N	22		22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	3.334		3.340		3.370		3.387		3.412
	SD	0.247		0.248		0.247		0.251		0.259
	N	22		22		22		22		22
	Trend	.		.		.		.		.
Sex: Female		Body Weight (Day of Gestation)
Group		23		24		25		26		27
Group: 1 Control 0 mg/kg/day	Mean	3.426		3.427		3.442		3.468		3.478
	SD	0.201		0.203		0.196		0.195		0.195
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Group: 2 m-xylene 30 mg/kg/day	Mean	3.449		3.446		3.463		3.483		3.490
	SD	0.249		0.253		0.243		0.243		0.242
	N	20		21		21		21		21
Group: 3 m-xylene 100 mg/kg/day	Mean	3.514		3.528		3.548		3.566		3.568
	SD	0.266		0.261		0.261		0.258		0.259
	N	22		22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	3.427		3.448		3.470		3.480		3.492
	SD	0.253		0.255		0.255		0.257		0.243
	N	22		22		22		22		22
	Trend	.		.		.		.		.
Sex: Female		Body Weight (Day of Gestation)
Group		28 (#)
Group: 1 Control 0 mg/kg/day	Mean	3.501
	SD	0.203
	N	21
	Trend	↑
Group: 2 m-xylene 30 mg/kg/day	Mean	3.511
	SD	0.244
	N	21
Group: 3 m-xylene 100 mg/kg/day	Mean	3.590
	SD	0.251
	N	22
Group: 4 m-xylene 300 mg/kg/day	Mean	3.522
	SD	0.250
	N	22
	Trend	> 0.05

Table 2 Body weight gains (kg) - group mean values

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		0 to 6 (#)		6 to 7 (#1)		6 to 8 (#1)		6 to 9 (#1)		6 to 10 (#1)		6 to 11 (#1)
Group: 1 Control 0 mg/kg/day	Mean	0.041		0.013		0.014		0.026		0.045		0.065
	SD	0.101		0.030		0.046		0.058		0.060		0.069
	N	21		21		21		21		21		21
	Trend	↑		↓		↓		↓		↓		↓
Group: 2 m-xylene 30 mg/kg/day	Mean	0.048		0.026		0.008		-0.008		-0.006		0.013
	SD	0.097		0.052		0.059		0.064		0.072		0.073
	N	21		21		21		21		21		21
	Trend	.		.		.		.		.		≤ 0.05	*
Group: 3 m-xylene 100 mg/kg/day	Mean	0.065		0.008		0.011		0.018		0.029		0.040
	SD	0.085		0.045		0.068		0.068		0.073		0.072
	N	22		22		22		22		22		22
	Trend	.		.		.		> 0.05		> 0.05		≤ 0.05	*
Group: 4 m-xylene 300 mg/kg/day	Mean	0.075		0.000		-0.016		-0.015		0.003		0.006
	SD	0.073		0.036		0.041		0.051		0.053		0.075
	N	22		22		22		22		22		22
	Trend	> 0.05		> 0.05		> 0.05		≤ 0.05	*	≤ 0.05	*	≤ 0.05	*
Sex: Female		Body Weight Gain  (Day of Gestation)
Group		6 to 12 (#)		6 to 13 (#)		6 to 14 (#)		6 to 15 (#1)		6 to 16 (#1)		6 to 17 (#1)
Group: 1 Control 0 mg/kg/day	Mean	0.090		0.123		0.161		0.186		0.198		0.219
	SD	0.076		0.087		0.084		0.092		0.099		0.097
	N	21		21		21		21		21		21
	Trend	↓		↓		↓		↓		↓		↓
Group: 2 m-xylene 30 mg/kg/day	Mean	0.042		0.075		0.107		0.132		0.144		0.150
	SD	0.073		0.070		0.070		0.078		0.088		0.094
	N	21		21		21		21		21		21
	Trend	.		.		≤ 0.05	*	.		.		.
Group: 3 m-xylene 100 mg/kg/day	Mean	0.066		0.092		0.122		0.155		0.174		0.176
	SD	0.069		0.075		0.079		0.082		0.089		0.083
	N	22		22		22		22		22		22
	Trend	> 0.05		> 0.05		≤ 0.05	*	.		.		.
Group: 4 m-xylene 300 mg/kg/day	Mean	0.027		0.064		0.095		0.120		0.142		0.146
	SD	0.094		0.099		0.108		0.120		0.129		0.137
	N	22		22		22		22		22		22
	Trend	≤ 0.05	*	≤ 0.05	*	≤ 0.05	*	> 0.05		> 0.05		> 0.05
Sex: Female		Body Weight Gain  (Day of Gestation)
Group		6 to 18 (#)		6 to 19 (#)		6 to 20 (#)		6 to 21 (#)		6 to 22 (#)		6 to 23 (#)
Group: 1 Control 0 mg/kg/day	Mean	0.232		0.246		0.266		0.284		0.302		0.312
	SD	0.099		0.096		0.098		0.096		0.104		0.103
	N	21		21		21		21		21		21
	Trend	↓		↓		↓		↓		↓		↓
Group: 2 m-xylene 30 mg/kg/day	Mean	0.165		0.190		0.212		0.231		0.249		0.275
	SD	0.098		0.101		0.094		0.106		0.114		0.106
	N	21		21		21		21		21		20
	Trend	.		.		.		.		.		.
Group: 3 m-xylene 100 mg/kg/day	Mean	0.195		0.201		0.231		0.253		0.281		0.296
	SD	0.086		0.091		0.088		0.081		0.078		0.072
	N	22		22		22		22		22		22
	Trend	> 0.05		> 0.05		> 0.05		> 0.05		.		.
Group: 4 m-xylene 300 mg/kg/day	Mean	0.166		0.172		0.202		0.220		0.245		0.259
	SD	0.126		0.127		0.116		0.117		0.123		0.117
	N	22		22		22		22		22		22
	Trend	≤ 0.05	*	≤ 0.05	*	≤ 0.05	*	≤ 0.05	*	> 0.05		> 0.05
Sex: Female		Body Weight Gain  (Day of Gestation)
Group		6 to 24 (#)		6 to 25 (#)		6 to 26 (#)		6 to 27 (#)		6 to 28 (#)
Group: 1 Control 0 mg/kg/day	Mean	0.313		0.329		0.354		0.365		0.388
	SD	0.118		0.113		0.115		0.126		0.130
	N	21		21		21		21		21
	Trend	↓		↓		↓		↓		↓
Group: 2 m-xylene 30 mg/kg/day	Mean	0.287		0.304		0.325		0.331		0.353
	SD	0.112		0.110		0.106		0.110		0.108
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Group: 3 m-xylene 100 mg/kg/day	Mean	0.310		0.330		0.348		0.350		0.372
	SD	0.075		0.068		0.075		0.078		0.079
	N	22		22		22		22		22
	Trend	.		.		.		.		.
Group: 4 m-xylene 300 mg/kg/day	Mean	0.280		0.302		0.312		0.324		0.354
	SD	0.120		0.122		0.131		0.128		0.133
	N	22		22		22		22		22
	Trend	> 0.05		> 0.05		> 0.05		> 0.05		> 0.05

(#) - Shirley, Kruskal-Wallis & Steel

(#1) - Williams, Anova & Dunnett: * = p ≤ 0.05

Table 3 Terminal Body Weights adjusted for gravis uterus weight

Sex: Female		Dead Body Weight  (kg)		Gravid Uterus Weight (g)		Body Weight Adjusted for Gravid  Uterus Weight (kg)		Adjusted Body Weight Gain (kg) (Day of Gestation)
Group		(#)		(#)		(#)		6 to 28 (#)
Group: 1 Control 0 mg/kg/day	Mean	3.50		432.2		3.06		-0.05
	SD	0.20		78.4		0.16		0.16
	N	21		21		21		21
	Trend	↑		↑		↑		↓
Group: 2 m-xylene 30 mg/kg/day	Mean	3.49		440.9		3.05		-0.11
	SD	0.24		82.8		0.23		0.14
	N	21		21		21		21
Group: 3 m-xylene 100 mg/kg/day	Mean	3.58		462.0		3.12		-0.10
	SD	0.25		67.0		0.22		0.13
	N	22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	3.50		427.0		3.07		-0.09
	SD	0.26		57.0		0.24		0.15
	N	22		22		22		22
	Trend	> 0.05		> 0.05		> 0.05		> 0.05

Table 4 Food intake (g/animal/day) group mean values

Sex: Female		Mean Food Intake  (Day of Gestation)
Group		4 to 5 (#)		5 to 6 (#)		6 to 8 (#)		8 to 10 (#1)		10 to 12 (#1)
Group: 1 Control 0 mg/kg/day	Mean	135.5		141.6		144.5		133.7		137.7
	SD	54.3		36.8		18.7		31.0		35.9
	N	21		21		20		20		20
	Trend	↑		↑		↓		↓		↓
Group: 2 m-xylene 30 mg/kg/day	Mean	139.9		153.4		147.4		121.5		129.0
	SD	59.3		27.9		26.1		36.1		37.4
	N	21		21		21		21		21
Group: 3 m-xylene 100 mg/kg/day	Mean	145.5		145.1		151.7		133.3		138.2
	SD	43.3		47.3		34.9		32.0		36.9
	N	22		22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	149.0		151.9		132.5		123.7		127.0
	SD	44.2		42.4		36.2		28.6		38.9
	N	22		22		22		21		21
	Trend	> 0.05		> 0.05		> 0.05		> 0.05		> 0.05
Sex: Female		Mean Food Intake  (Day of Gestation)
Group		12 to 14 (#)		14 to 16 (#)		16 to 18 (#1)		18 to 20 (#1)		20 to 22 (#1)
Group: 1 Control 0 mg/kg/day	Mean	147.4		137.4		132.1		153.2		147.6
	SD	27.3		28.7		34.9		37.5		27.0
	N	20		21		21		21		21
	Trend	↑		↑		↓		↓		↓
Group: 2 m-xylene 30 mg/kg/day	Mean	150.4		134.4		115.2		144.2		150.0
	SD	33.0		38.4		37.2		34.3		36.6
	N	21		21		21		21		21
Group: 3 m-xylene 100 mg/kg/day	Mean	149.9		142.3		131.3		146.7		148.5
	SD	24.4		31.9		35.3		42.6		26.8
	N	22		22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	146.5		134.9		125.8		147.2		142.0
	SD	37.7		47.4		39.6		32.5		26.2
	N	21		22		21		21		21
	Trend	> 0.05		> 0.05		> 0.05		> 0.05		> 0.05
Sex: Female		Mean Food Intake  (Day of Gestation)						Overall Mean Food Intake (Day of Gestation)
Group		22 to 24 (#)		24 to 26 (#1)		26 to 28 (#)		6 to 28 (#1)
Group: 1 Control 0 mg/kg/day	Mean	113.6		108.7		104.9		132.0
	SD	34.3		46.4		24.4		16.8
	N	21		21		21		20
	Trend	↑		↑		↑		↑
Group: 2 m-xylene 30 mg/kg/day	Mean	129.0		122.2		111.8		131.5
	SD	28.0		38.3		33.2		25.0
	N	21		21		20		20
Group: 3 m-xylene 100 mg/kg/day	Mean	125.8		110.1		97.8		134.1
	SD	25.3		26.1		24.6		22.8
	N	22		22		22		22
Group: 4 m-xylene 300 mg/kg/day	Mean	129.6		117.1		113.0		132.8
	SD	23.5		33.4		35.9		20.5
	N	22		22		22		20
	Trend	> 0.05		> 0.05		> 0.05		> 0.05

(#) - Williams, Anova & Dunnett

(#1) - Williams, Anova & Dunnett(Log)

Table 5 Pregnancy Data Summary

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 m-xylene 30 mg/kg/day		Group: 3 m-xylene 100 mg/kg/day		Group: 4 m-xylene 300 mg/kg/day
Group Size		22		22		22		22
Not Pregnant		1		1		0		0
Not Pregnant %		4.5		4.5		0.0		0.0
Not Pregnant Died/Killed	Sum	0		0		0		0
Not Pregnant Schedule Kill	Sum	1		1		0		0
Pregnant		21		21		22		22
Pregnant %		95.5		95.5		100.0		100.0
Pregnant Died/Killed/Aborted	Sum	0		0		0		0
Pregnant with Total Resorption	Sum	0		0		0		0
Number with Live Foetuses	Sum	21		21		22		22

Table 6 Uterine and Implantation Data - Group Mean Values

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 m-xylene 30 mg/kg/day		Group: 3 m-xylene 100 mg/kg/day		Group: 4 m-xylene 300 mg/kg/day
Number with Live Foetuses (GD 28)		21		21		22		22
Number of Corpora Lutea (#)	Sum	206		204		220		208
	Mean	9.8		9.7		10.0		9.5
	SD	1.7		1.8		1.5		1.5
	Trend	↓		.		.		> 0.05
Number of Implantations (#1)	Sum	182		187		209		194
	Mean	8.7		8.9		9.5		8.8
	SD	1.8		2.2		1.4		1.3
	Trend	↑		.		.		> 0.05
% Pre-implantation Loss (#2)	Mean	11.4		8.7		4.8		6.4
	Trend	↓		.		.		> 0.05
Number of Early Deaths (#2)	Sum	4		4		3		8
	Mean	0.2		0.2		0.1		0.4
	SD	0.5		0.4		0.4		0.5
	Trend	↑		.		.		> 0.05

(#) - Williams, Anova & Dunnett(Log)

(#1) - Williams, Anova & Dunnett

(#2) - Shirley, Kruskal-Wallis & Steel

Table 6 Uterine and Implantation Data - Group Mean Values (Continued)

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 m-xylene 30 mg/kg/day		Group: 3 m-xylene 100 mg/kg/day		Group: 4 m-xylene 300 mg/kg/day
Number of Late Deaths (#)	Sum	0		4		2		0
	Mean	0.0		0.2		0.1		0.0
	SD	0.0		0.9		0.4		0.0
	Trend	↑		.		.		> 0.05
Number of Dead Foetuses	Sum	0		0		0		0
	Mean	0.0		0.0		0.0		0.0
	SD	0.0		0.0		0.0		0.0
Number of Live Foetuses (#1)	Sum	178		179		204		186
	Mean	8.5		8.5		9.3		8.5
	SD	1.8		1.9		1.4		1.3
	Trend	↑		.		.		> 0.05
% Post-implantation Loss (#)	Mean	2.0		3.7		2.3		4.1
	Trend	↑		.		.		> 0.05
Live Foetuses as % of Implants	Mean	98.0		96.3		97.7		95.9

(#) - Shirley, Kruskal-Wallis & Steel

(#1) - Williams, Anova & Dunnett

Table 7 Litter Weights (g) Foetal Data - Group Mean Values

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 m-xylene 30 mg/kg/day		Group: 3 m-xylene 100 mg/kg/day		Group: 4 m-xylene 300 mg/kg/day
Number with Live Foetuses		21		21		22		22
No of Live Foetuses		178		179		204		186
No of Male Foetuses		86		85		99		93
No of Female Foetuses		92		94		105		93
% of Male Foetuses (#)	Mean	49.5		49.0		47.7		49.8
	Trend	↓		.		.		> 0.05
Litter Weight (g) (#)	Mean	295.74		292.78		318.27		294.68
	Trend	↑		.		.		> 0.05
Foetal Weight (M+F) (g) (#)	Mean	35.29		34.84		34.51		35.04
	Trend	↓		.		.		> 0.05
Foetal Weight (M) (g)	Mean	35.64		35.24		35.11		35.51
Foetal Weight (F) (g)	Mean	34.99		34.55		34.12		34.50
Placental Weight (g) (#)	Mean	3.63		3.62		3.57		3.61
	Trend	↓		.		.		> 0.05

#) - Williams, Anova & Dunnett

Data are recorded and reported against dams, day of lactation is equivalent to day of age

Table 8 Examination of Foetuses - Summary of Group Mean Values

	Control	Group 2	Group 3	Group 4
Dose Level mg/kg bw/day :	0	30	100	300
Total Number of Litters Examined:	21	21	22	22
Total Number of Foetuses Examined:	178	179	204	186
Number with Major Abnormalities:	6	5	2	5
Mean % of Foetuses Examined	3.1	2.9	0.9	2.8
Number of Litters Affected#	5	4	2	4
Number with Minor Abnormalities:	79	71	111	85
Mean % of Foetuses Examined	45.3	40.8	52.8	45.8
Number of Litters Affected#	21	20	21	22
Number with Variations:	177	179	204	186
Mean % of Foetuses Examined	99.6	100.0	100.0	100.0
Number of Litters Affected#	21	21	22	22

# Statistically Analysed

Table 9 Examination of Foetuses - Group Mean Values - by foetus

		Control	Group 2	Group 3	Group 4
Dose Level mg/kg bw/day :		0	30	100	300
	Number of foetuses affected (group mean %)
Total Number of Litters Examined:		21	21	22	22
Total Number of Foetuses Examined:		178	179	204	186
Key Finding#:	Type
Head
A: jaw: cheiloschisis	major	0(0.0)	1(0.7)	0(0.0)	0(0.0)
Brain
B exencephaly	major	0(0.0)	0(0.0)	1(0.3)	0(0.0)
Oral Cavity
la tongue-tip: bifurcated	minor	0(0.0)	1(0.7)	0(0.0)	0(0.0)
C tongue-tip: ankyloglossia	major	0(0.0)	2(1.4)	0(0.0)	0(0.0)
Umbilicus
D umbilicus:omphalocele	major	0(0.0)	0(0.0)	0(0.0)	1(0.5)
Hindlimb
1b hindpaw uni-or bilateral:abnormal flexure slight	minor	0(0.0)	1(0.7)	0(0.0)	0(0.0)
E digits - uni- or bilateral:polydactyly	major	0(0.0)	2(1.4)	0(0.0)	0(0.0)
# statistically analysed
Neck
lc trachea: indistinct cartilage rings	minor	1(0.5)	0(0.0)	0(0.0)	0(0.0)
Thoracic Cavity
1d subclavian artery -right: retro- oesophageal	minor	0(0.0)	0(0.0)	2(0.9)	3J (1.4)
a:common carotid artery- left: arising from innominate artery	variant	18(9.4)	22(12.3)	16(7.8)	25(13.5)
le aortic arch: enlarged slight	minor	0(0.0)	0(0.0)	1(0.5)	0(0.0)
F aortic arch: enlarged severe	major	0(0.0)	1(0.5)	0(0.0)	0(0.0)
G aortic arch: retro-oesophageal	major	1(0.5)	0(0.0)	0(0.0	1(0.5)
b aortic arch: additional blood vessel	variant	8(4.5)	19(10.5)	3(1.3)	5(2.7)
lf pulmonary arch:narrowed slight	minor	0(0.0)	0(0.0)	1(0.5)	0(0.0)
H pulmonary arch:narrowed severe	major	0(0.0)	1(0.5)	0(0.0)	0(0.0)
I aortic and pulmonary arch: transposition of great vessels	major	0(0.0)	0(0.0)	0(0.0)	1(0.5)
lg ductus arteriosus: narrowed slight	minor	0(0.0)	1(0.5)	1(0.5)	0(0.0)
1h ventricle uni-or bilateral: reduced in size slight	minor	0(0.0)	0(0.0)	1(0.5)	1(0.5)
#statistically analysed
J = p<0.05 (Jonckheere Test)
Thoracic Cavity (continued)
1i ventricle uni-or bilateral: enlarged slight	minor	0(0.0)	0(0.0)	0(0.0)	1(0.5)
J interventricular septum: incomplete	major	0(0.0)	1(0.5)	0(0.0)	1(0.5)
1j pericardial sac: fluid filled	minor	0(0.0)	1(0.7)	0(0.0)	0(0.0)
1k thymus- 1 or more lobe: undescended	minor	4(2.3)	3(1.7)	2(1.1)	1(0.6)
1l post caval lung lobe:absent	minor	4(2.0)	19(10.4)	23(11.1)	25JJ(13.3)
Number of litters affected		4	9	8	13C
Abdominal Cavity
1m kidney uni- or bilateral:increased pelvic cavitation	minor	0(0.0)	2(1.2)	0(0.0)	0(0.0)
1n ovary uni-or bilateral: cystic	minor	1(0.6)	2(1.0)	2(1.0)	0(0.0)
1o umbilical artery: thickened	minor	1(0.6)	0(0.0)	0(0.0)	0(0.0)
1p liver 1 or more lobe: accessory lobe present	minor	16(10.9)	4(2.5)	6(2.9)	6(3.8)
1q gall bladder: reduced in size	minor	2(1.2)	0(0.0)	1(0.8)	4(2.4)
1r gall bladder: not detected	minor	1(0.6)	0(0.0)	1(0.5)	0(0.0)
1s gall bladder: bilobed	minor	1(0.6)	0(0.0)	0(0.0)	0(0.0)
#statistically analysed
C= p<0.05 (Cochran-Armitage Test)
JJ = p<0.01 (Jonckheere Test)
Total Number of Litters Examined:		21	21	22	22
Total Number of Foetuses Examined:		88	90	102	94
Brain
1u lateral ventricle: enlarged slight	minor	0(0.0)	2(2.4)	0(0.0)	0(0.0)
# statistically analysed

Table 9 Examination of Foetuses - Group Mean Values - by foetus (Continued)

Key finding	Type	Control	Group 2	Group 3	Group 4
Thoracic cavity(total number of foetuses examined (total number of litters examined))		178 (21)	179 (21)	204 (22)	186 (22)
subclavian artery- right: retro-oesophageal	minor	0(0.0)	0(0.0)	2(0.9)	3J(1.4)
post caval lung lobe: absent	minor	4(2.0)	19(10.4)	23(11.1)	25JJ(13.3)
Skull(total number of foetuses examined (total number of litters examined))		88 (21)	90 (21)	102 (22)	94 (22)
one or more: fissure/plaque of bone integral to normal structure of bone	variant	0(0.0)	3(3.6)	1(1.1)	4J(4.3)
Rib(total number of foetuses examined (total number of litters examined))		178 (21)	179 (21)	204 (22)	186 (22)
13th- uni- or bilateral: extra	variant	79(44.5)	79(45.4)	90(43.4)	115J(62.5)

Number of foetuses affected (group mean percent)

J = p<0.05; JJ = p<0.05; JJJ = p<0.001 (Jonckheere Test)

Conclusions:

Administration of m-xylene to pregnant New Zealand White rabbits once daily by oral gavage, from Day 6 of gestation until Day 27 of gestation inclusive, elicited slight maternal toxicity at 300 mg/kg/day in terms of transient post-dose observations of purple ears and rapid breathing and initial body weight loss, with no effect on overall body weight gain or food intake. There was no adverse effect on pregnancy, embryonic or foetal development at any dose level. 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.

Executive summary:

The study objective of this study was to investigate the effects of the test item, m-xylene, on the pregnant New Zealand White rabbit and developing organism when administered from Day 6 to Day 27 of gestation, inclusive. Four groups of 22 sexually mature, timed-mated, female New Zealand White rabbits were given m-xylene by gavage, at 0 (vehicle Control), 30, 100 or 300 mg/kg/day once daily at a dose volume of 5 mL/kg body weight from Day 6 to Day 27 of gestation, inclusive. All females were observed daily from the start of dosing and body weight and food intake were recorded at regular intervals. The animals were killed on Day 28 of gestation, a necropsy performed and the internal organs were examined for macroscopic abnormalities. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral and skeletal (bone and cartilage) abnormalities and variants. Approximately 50 % of the foetuses in each litter were decapitated and fixed in Bouin’s fluid for subsequent fixed head examination. There were no deaths during the study. Clinical signs were limited to abnormally coloured ears (purple), observed in all dose groups with the highest incidence at 300 mg/kg/day, accompanied by rapid breathing at 300 mg/kg/day. These clinical signs were present immediately after dosing but were not observed after one hour. At 300 mg/kg/day, there was a mean body weight loss between Days 6 and 9 of gestation; thereafter, body weight gain was similar to, or greater than Control, so that overall body weight gain was similar in all groups. There was no adverse effect on food intake. There was no effect on the number of implantations, incidence of pre- or post-implantation loss or on the number of live foetuses. There was no effect of m-xylene on foetal or placental weights or on the foetal sex ratio. The nature, incidence and inter-group distribution of visceral and skeletal foetal abnormalities and variations did not indicate an adverse effect of m-xylene. Administration of m-xylene to pregnant New Zealand White rabbits once daily by oral gavage, from Day 6 of gestation until Day 27 of gestation inclusive, elicited slight maternal toxicity at 300 mg/kg/day in terms of transient post-dose observations of purple ears and rapid breathing and initial body weight loss, with no effect on overall body weight gain or food intake. There was no adverse effect on pregnancy, embryonic or foetal development at any dose level. 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.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd October 2019 - 3rd December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25th June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

o-xylene
Batch number: STBH7551
Appearance: Clear colourless liquid
Retest date (5 years after opening): 17 September 2024 and 18 October 2024
Quantity supplied: 7 x 1 litre
Purity: 99.0 % (GC area %)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Specification
Eighty-eight timed-mated female New Zealand White rabbits were supplied by Envigo Global Services Inc., 310 Swampbridge Road, Denver, PA 17517, USA.
Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.
The animals were approximately five months of age on arrival and on examination were found to be healthy. After at least three days acclimatisation, they were re-examined and confirmed to be suitable for use. On the first day of dosing the females weighed 2.82 to 3.88 kg.

Allocation and identification
Allocation to groups was performed using a stratified randomisation procedure based on individual body weights recorded on Day 0 of gestation at the suppliers (making sure that, where possible, females mated with the same male were spread across the groups). Each animal was uniquely identified by a subcutaneously implanted micro-identification device. To aid identification during procedures, where required, the animals were ear marked with their own unique identification numbers using an indelible pen. This mark was placed on the opposite ear to that of the supplier's number.

Housing and environmental conditions
The animals were housed individually in perforated-floor cages suspended over paper-lined trays. The study room was illuminated to give a cycle of 12 hours light and 12 hours dark and was air-conditioned. The target ranges for temperature and humidity were 16 °C to 20 °C and 40 % to 70 %, respectively. Recorded values were within, or marginally outside, these limits. The only deviation was that, on the 16 to 17 October 2019, the temperature was at a maximum of 23 °C. As the recorded temperature was not outside of the specified range for a long period of time and as the health of the animals was not affected, this deviation was considered not to have affected the outcome or integrity of the study.

Diet and water
A pelleted diet, Teklad 2030C supplied by Envigo RMS (UK) Limited and mains tap water were freely available. From the day of arrival, approximately 30 g of hay was provided each day to animals as enrichment. Hay was supplied by IPS Product Supplies Ltd., BCM IPS Ltd., London, WC1N 3XX, United Kingdom. Where considered necessary, when food intake was reduced, feeding regimens were adjusted to encourage and maintain gut motility; details of the additional diet, carrots and moistened diet offered. Flavoured Bunny Blocks™ (supplied by LBS Servicing Biotechnology Ltd, PO Box 431, RH6 0UW, United Kingdom) were available and replaced each week. Certificates of analysis for pelleted diet and water are held centrally and retained within the Sequani archives. It was considered that none of the contaminants that were monitored was present at a level that might have prevented the study objectives from being achieved.

Route of administration:

oral: gavage

Vehicle:

other: 1% methyl cellulose w/v, 1% Tween 80 v/v

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentrations of o-xylene in test formulations prepared on 02 October 2019 for the first week of dosing ranged from -19.7 % for the nominal 60 mg/mL level down to -76.7 % for the nominal 6 mg/mL level. Replication of the duplicate samples and procedural recoveries were satisfactory. As a result of the high deviation from nominal, contingency samples (10 mL) from the mix were supplied and analyzed. The relative mean error of the analysed concentrations ranged from -16.5 % for the nominal 60 mg/mL level down to -67.0 % for the nominal 6 mg/mL level. Replication of the duplicate samples and procedural recoveries were satisfactory. The mean concentrations and homogeneity of o-xylene in test formulations prepared on 14 October 2019 ranged from -6.3 % for the nominal 60 mg/mL level down to -37.3 % for the nominal 6 mg/mL level. Homogeneity of the formulations measured as CV% of the six samples at each level and procedural recoveries were satisfactory. Following discussions between the Study Director and Principal Investigator it was thought that the low analysed concentrations might be due to losses of the volatile test item to the headspace in the sample vials. For that reason samples from test formulations prepared on 23 October 2019 and 29 October 2019 were supplied for analysis in 2 mL vials in order to minimize the headspace. Additional samples from the 29 October 2019 formulations were supplied for analysis in 8 mL vials. The concentrations of o-xylene in test formulations prepared on 23 October 2019 ranged from -5.7 % for the nominal 60 mg/mL level down to -10.0 % for the nominal 6 mg/mL level. Procedural recoveries were satisfactory. The concentrations of o-xylene in test formulations prepared on 29 October 2019 supplied in 2 mL vials ranged from -6.0% for the nominal 60 mg/mL level down to -9.3 % for the nominal 6 mg/mL level, whereas for the samples supplied in 8 mL vials ranged from -7.8 % for the nominal 60 mg/mL level down to -41.0 % for the nominal 6 mg/mL level. Procedural recoveries were satisfactory. This clearly demonstrates that o xylene losses to the headspace are considerable when samples are supplied in the 8 mL vials. The mean concentrations for each formulation prepared on 23 October 2019 and 29 October 2019 were within ±10 % of the nominal concentration, confirming the accuracy of formulation. No detectable concentrations of o xylene were found in any control formulation samples.

Details on mating procedure:

Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.

Duration of treatment / exposure:

OECD 414 Main study. From Day 6 to Day 27 of gestation, inclusive.

Frequency of treatment:

Once Daily

Duration of test:

From Day 6 to Day 28 of gestation

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

A total of 88 animals were used in this study, the design of which is in the Table 1 below in Any Other Information. Animals were dosed once daily from Day 6 to Day 27 of gestation, inclusive, by gavage, using a rubber catheter and disposable syringe at a constant dose volume of 5 mL/kg body weight. Individual doses were adjusted according to the most recent body weight.

Maternal examinations:

Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily. From the start of dosing, animals were observed before and shortly after dosing. On weekdays, animals were also observed at one hour after dosing and at the weekend they were observed either at one hour after dosing or at the end of the working day, whichever was sooner.

Necropsy
The females were killed on Day 28 of gestation by an overdose injection of sodium pentobarbitone. The animals were weighed, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained in 70 % IDA (industrial denatured alcohol) for approximately seven days and then transferred and retained in 10 % buffered formalin.

Ovaries and uterine content:

For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix. The foetuses and their placentae were removed and the uterus and ovaries retained in 10 % buffered formalin.

Fetal examinations:

On Day 28 of gestation, live foetuses were killed by an intraperitoneal injection of sodium pentobarbitone solution, weighed and examined for external abnormalities.
Approximately 50 % of the foetuses in each litter were decapitated and the heads fixed in Bouin's solution for subsequent serial sectioning. The intact foetuses and the bodies of the decapitated foetuses were placed in 70 % IDA. Later in the day they were skinned, dissected, the viscera were examined and the sex recorded. They were eviscerated and then a coronal section was made through the head, along the frontal parietal suture and the brain examined. The carcasses were then cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined.
Structural congenital abnormalities that impair, or potentially impair, the survival or constitution of the foetus were classified as major abnormalities. Abnormalities that in isolation, do not impair the survival or constitution of the foetus but could potentially interfere with normal bodily function were classified as minor abnormalities. Alternative structures occurring regularly in the control population, which may be permanent or transient, were classified as variants.
Foetuses with major external or visceral abnormalities were photographed.
For archiving, all foetal heads fixed in Bouin's fluid were stored in 10 % buffered formalin and all skeletal specimens were stored in aqueous glycerol with thymol crystals (to prevent fungal growth).

Statistics:

Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1. Depending on the nature of the data set that was to be analysed, appropriate tests were applied, as indicated in the table below (Table 2 in Any Other Information). Where parametric tests were appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied. Proportions of foetuses affected were treated as continuous non-parametric data, using one-sided step-wise Jonckheere Tests. Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.

Indices:

Pre-implantation loss (%) = (no. of corpora lutea – no. of implantation sites)/ no. of corpora lutea x 100
Post-implantation loss (%) = (no. of implantation sites – no. of live foetuses)/ no. of implantation sites x 100

Mean pre- and post-implantation losses were calculated on a proportional litter basis. Mean foetal body weights were calculated separately by sex for each litter; group mean body weights were calculated (separately by sex) from the litter means. The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.

Historical control data:

Historical control data available see attached 'background material'.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, pink/purple ears and rapid breathing were observed in most animals throughout the dosing period. These observations were seen immediately after dosing but were no longer present at one hour post-dose. Rapid breathing and purple ears were observed immediately after dosing of five females given 100 mg/kg/day and six females given 30 mg/kg/day, between Days 10 and 13 of gestation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no deaths during the study that were associated with o-xylene. One Control animal was killed on Day 15 of gestation following inappetence from arrival and marked body weight loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, overall mean body weight gain was statistically significantly(p≤0.05) lower than Controls. Generally, animals in this group lost weight over the first three days of dosing (Days 6 to 9 of gestation) and group mean body weight gains were lower than Controls until approximately Day 19 of gestation and then similar to, or slightly higher than, Controls. There were no effects on body weight or body weight gain at 30 or 100 mg/kg/day. Terminal body weight adjusted for the weight of the gravid uterus was similar in all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, overall mean food intake was significantly lower than Control (p≤0.001). At 100 mg/kg/day, overall mean food intake was slightly lower than Control (9 %) and statistically significantly lower on occasion (p≤0.05, p≤0.01). There was no effect on food intake at 30 mg/kg/day. One female given 30 mg/kg/day and four females given 300 mg/kg/day, were offered moistened diet to encourage eating following a decline in food intake during the dosing period. In addition, one Control female, one given 100 mg/kg/day and one given 300 mg/kg/day, did not eat from arrival and were also offered moistened diet.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

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:

no effects observed

Description (incidence and severity):

There were no findings at maternal necropsy associated with o-xylene.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no effects on the pregnancy parameters that were attributed to o-xylene. At 300 mg/kg/day, the mean number of live foetuses per litter was marginally lower than Controls; this was a consequence of slightly fewer implantations in this group compared with Control and all parameters remained within the historical Control ranges.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no effects on the pregnancy parameters that were attributed to o-xylene. At 300 mg/kg/day, the mean number of live foetuses per litter was marginally lower than Controls; this was a consequence of slightly fewer implantations in this group compared with Control and all parameters remained within the historical Control ranges.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no effects on the pregnancy parameters that were attributed to o-xylene. At 300 mg/kg/day, the mean number of live foetuses per litter was marginally lower than Controls; this was a consequence of slightly fewer implantations in this group compared with Control and all parameters remained within the historical Control ranges.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no effects on the pregnancy parameters that were attributed to o-xylene.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no effects on the pregnancy parameters that were attributed to o-xylene.

Other effects:

not examined

Details on maternal toxic effects:

There were no deaths during the study that were associated with o-xylene. One Control animal was killed on Day 15 of gestation following inappetence from arrival and marked body weight loss.
At 300 mg/kg/day, pink/purple ears and rapid breathing were observed in most animals throughout the dosing period. These observations were seen immediately after dosing but were no longer present at one hour post-dose. Rapid breathing and purple ears were observed immediately after dosing of five females given 100 mg/kg/day and six females given 30 mg/kg/day, between Days 10 and 13 of gestation.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of o-xylene administration on mean foetal weight. At 300 mg/kg/day, mean litter weight was slightly lower than Control (p ≤ 0.05) due to the slightly smaller litter size.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no effects on mean foetal sex ratio or placental weights. control group: 98 male, 88 female offspring, 30mg/kg/day group: 97 male 99 female offspring; 100 mg/kg/day group: 83 male 102 female offspring; 300 mg/kg bw/ day group: 80 male 92 female offspring.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

At 300 mg/kg/day, mean litter weight was slightly lower than Control (p ≤ 0.05) due to the slightly smaller litter size. There was no effect of o-xylene administration on mean foetal weight.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

No effects associated with o-xylene administration

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

Key result

Developmental effects observed:

no

Treatment related:

no

Table 1 Body weights (kg) - Group Mean Values

Sex: Female		Body Weight (Day of Gestation)
Group		0		4		5		6 (#)		7
Group: 1 Control 0 mg/kg/day	Mean	3.287		3.248		3.278		3.316		3.312
	SD	0.229		0.263		0.265		0.272		0.273
	N	21		21		21		21		21
	Trend	.		.		.		↑		.
Group: 2 o-xylene 30 mg/kg/day	Mean	3.325		3.267		3.328		3.356		3.377
	SD	0.259		0.243		0.237		0.235		0.237
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	3.271		3.225		3.269		3.290		3.291
	SD	0.245		0.212		0.218		0.238		0.228
	N	21		21		21		21		21
Group: 4 o-xylene 300 mg/kg/day	Mean	3.335		3.301		3.328		3.353		3.329
	SD	0.266		0.263		0.281		0.287		0.275
	N	21		21		21		21		21
	Trend	.		.		.		> 0.05		.
Sex: Female		Body Weight (Day of Gestation)
Group		8		9		10		11		12
Group: 1 Control 0 mg/kg/day	Mean	3.330		3.336		3.344		3.356		3.385
	SD	0.278		0.281		0.280		0.285		0.292
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Group: 2 o-xylene 30 mg/kg/day	Mean	3.375		3.383		3.394		3.406		3.439
	SD	0.247		0.248		0.250		0.245		0.244
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	3.302		3.310		3.328		3.344		3.371
	SD	0.212		0.208		0.206		0.203		0.212
	N	21		21		21		21		21
Group: 4 o-xylene 300 mg/kg/day	Mean	3.314		3.310		3.302		3.317		3.332
	SD	0.271		0.274		0.272		0.280		0.282
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Sex: Female		Body Weight (Day of Gestation)
Group		13		14		15		16		17
Group: 1 Control 0 mg/kg/day	Mean	3.398		3.429		3.477		3.527		3.534
	SD	0.293		0.288		0.291		0.273		0.272
	N	21		21		21		20		20
	Trend	.		.		.		.		.
Group: 2 o-xylene 30 mg/kg/day	Mean	3.461		3.488		3.510		3.523		3.548
	SD	0.234		0.231		0.242		0.248		0.257
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	3.391		3.418		3.450		3.469		3.473
	SD	0.216		0.221		0.227		0.225		0.213
	N	21		21		21		21		21
Group: 4 o-xylene 300 mg/kg/day	Mean	3.367		3.394		3.421		3.433		3.431
	SD	0.271		0.264		0.262		0.256		0.246
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Sex: Female		Body Weight (Day of Gestation)
Group		18		19		20		21		22
Group: 1 Control 0 mg/kg/day	Mean	3.546		3.568		3.590		3.624		3.639
	SD	0.279		0.280		0.289		0.291		0.288
	N	20		20		20		20		20
	Trend	.		.		.		.		.
Group: 2 o-xylene 30 mg/kg/day	Mean	3.561		3.577		3.599		3.618		3.631
	SD	0.254		0.261		0.260		0.258		0.265
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	3.482		3.507		3.532		3.555		3.568
	SD	0.210		0.209		0.210		0.217		0.214
	N	21		21		21		21		21
Group: 4 o-xylene 300 mg/kg/day	Mean	3.429		3.440		3.461		3.481		3.508
	SD	0.250		0.244		0.236		0.238		0.234
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Sex: Female		Body Weight (Day of Gestation)
Group		23		24		25		26		27
Group: 1 Control 0 mg/kg/day	Mean	3.656		3.661		3.656		3.676		3.675
	SD	0.282		0.274		0.268		0.254		0.253
	N	20		20		20		20		20
	Trend	.		.		.		.		.
Group: 2 o-xylene 30 mg/kg/day	Mean	3.648		3.652		3.651		3.659		3.673
	SD	0.266		0.265		0.259		0.247		0.250
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	3.573		3.572		3.571		3.570		3.583
	SD	0.219		0.229		0.220		0.218		0.217
	N	21		21		21		21		21
Group: 4 o-xylene 300 mg/kg/day	Mean	3.522		3.527		3.548		3.560		3.575
	SD	0.235		0.222		0.213		0.212		0.205
	N	21		21		21		21		21
	Trend	.		.		.		.		.
Sex: Female		Body Weight (Day of Gestation)
Group		28 (#)
Group: 1 Control 0 mg/kg/day	Mean	3.694
	SD	0.252
	N	20
	Trend	↓
Group: 2 o-xylene 30 mg/kg/day	Mean	3.701
	SD	0.258
	N	22
Group: 3 o-xylene 100 mg/kg/day	Mean	3.599
	SD	0.211
	N	21
Group: 4 o-xylene 300 mg/kg/day	Mean	3.606
	SD	0.206
	N	21
	Trend	> 0.05

(#) - Williams, Anova & Dunnett

Table 2 Body weight gains (kg) - group mean values

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		0 to 6 (#)		6 to 7 (#)		6 to 8 (#)		6 to 9 (#)		6 to 10 (#1)
Group: 1 Control 0 mg/kg/day	Mean	0.029		-0.004	**	0.014		0.020		0.029
	SD	0.132		0.058		0.086		0.087		0.078
	N	21		21		21		21		21
	Trend	↓		↓		↓		↓		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	0.031		0.021		0.019		0.027		0.038
	SD	0.088		0.038		0.053		0.061		0.065
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	0.019		0.002		0.013		0.021		0.038
	SD	0.106		0.050		0.063		0.079		0.090
	N	21		21		21		21		21
	Trend	.		.		> 0.05		> 0.05		> 0.05
Group: 4 o-xylene 300 mg/kg/day	Mean	0.018		-0.025		-0.039		-0.044		-0.051
	SD	0.127		0.033		0.049		0.074		0.073
	N	21		21		21		21		21
	Trend	> 0.05		> 0.05		≤ 0.05	*	≤ 0.05	*	≤ 0.01	**

(#) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05; ** = p ≤ 0.01

 (#1) - Williams, Anova & Dunnett: ** = p ≤ 0.01

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		6 to 11 (#)		6 to 12 (#)		6 to 13 (#)		6 to 14 (#1)		6 to 15 (#)
Group: 1 Control 0 mg/kg/day	Mean	0.040		0.069		0.082		0.113	**	0.161
	SD	0.073		0.100		0.101		0.114		0.106
	N	21		21		21		21		21
	Trend	↓		↓		↓		↓		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	0.050		0.083		0.105		0.132		0.154
	SD	0.073		0.057		0.064		0.062		0.076
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	0.054		0.081		0.102		0.128		0.160
	SD	0.097		0.091		0.091		0.091		0.103
	N	21		21		21		21		21
	Trend	> 0.05		> 0.05		> 0.05		.		> 0.05
Group: 4 o-xylene 300 mg/kg/day	Mean	-0.036		-0.021		0.014		0.041		0.068
	SD	0.079		0.078		0.081		0.092		0.103
	N	21		21		21		21		21
	Trend	≤ 0.01	**	≤ 0.001	***	≤ 0.05	*	> 0.05		≤ 0.01	**

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

(#1) - Shirley, Kruskal-Wallis & Steel: ** = p ≤ 0.01

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		6 to 16 (#)		6 to 17 (#)		6 to 18 (#)		6 to 19 (#)		6 to 20 (#)
Group: 1 Control 0 mg/kg/day	Mean	0.187		0.194		0.206		0.228		0.250
	SD	0.107		0.109		0.108		0.102		0.109
	N	20		20		20		20		20
	Trend	↓		↓		↓		↓		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	0.167		0.192		0.205		0.221		0.243
	SD	0.068		0.091		0.094		0.097		0.102
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	0.179		0.183		0.192		0.218		0.242
	SD	0.090		0.093		0.103		0.114		0.120
	N	21		21		21		21		21
	Trend	> 0.05		> 0.05		> 0.05		> 0.05		> 0.05
Group: 4 o-xylene 300 mg/kg/day	Mean	0.080		0.078		0.075		0.086		0.108
	SD	0.114		0.114		0.104		0.103		0.101
	N	21		21		21		21		21
	Trend	≤ 0.001	***	≤ 0.001	***	≤ 0.001	***	≤ 0.001	***	≤ 0.001	***

(#) - Williams, Anova & Dunnett: *** = p ≤ 0.001

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		6 to 21 (#)		6 to 22 (#)		6 to 23 (#)		6 to 24 (#)		6 to 25 (#)
Group: 1 Control 0 mg/kg/day	Mean	0.283		0.299		0.315		0.320		0.316
	SD	0.126		0.130		0.125		0.119		0.116
	N	20		20		20		20		20
	Trend	↓		↓		↓		↓		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	0.262		0.275		0.292		0.296		0.295
	SD	0.110		0.113		0.104		0.100		0.091
	N	22		22		22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	0.266		0.279		0.284		0.283		0.282
	SD	0.120		0.133		0.131		0.135		0.138
	N	21		21		21		21		21
	Trend	> 0.05		> 0.05		> 0.05		> 0.05		> 0.05
Group: 4 o-xylene 300 mg/kg/day	Mean	0.128		0.154		0.169		0.174		0.195
	SD	0.101		0.101		0.103		0.116		0.122
	N	21		21		21		21		21
	Trend	≤ 0.001	***	≤ 0.001	***	≤ 0.001	***	≤ 0.001	***	≤ 0.01	**

(#) - Williams, Anova & Dunnett: ** = p ≤ 0.01; *** = p ≤ 0.001

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		6 to 26 (#)		6 to 27 (#)		6 to 28 (#)
Group: 1 Control 0 mg/kg/day	Mean	0.335		0.335		0.353
	SD	0.127		0.129		0.115
	N	20		20		20
	Trend	↓		↓		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	0.303		0.317		0.345
	SD	0.079		0.076		0.085
	N	22		22		22
Group: 3 o-xylene 100 mg/kg/day	Mean	0.280		0.294		0.309
	SD	0.146		0.151		0.156
	N	21		21		21
	Trend	> 0.05		> 0.05		> 0.05
Group: 4 o-xylene 300 mg/kg/day	Mean	0.207		0.222		0.252
	SD	0.130		0.143		0.144
	N	21		21		21
	Trend	≤ 0.01	**	≤ 0.01	**	≤ 0.05	*

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

Table 3 Terminal body weight (kg) and weight gain (kg), adjusted for gravid uterus weight (g)- group mean values

Sex: Female		Dead Body Weight  (kg)		Gravid Uterus Weight (g)		Body Weight Adjusted for Gravid  Uterus Weight (kg)		Adjusted Body Weight Gain (kg) (Day of Gestation)
Group		(#)		(#)		(#)		6 to 28 (#)
Group: 1 Control 0 mg/kg/day	Mean	3.68		461.2		3.22		-0.12
	SD	0.25		77.5		0.22		0.12
	N	20		20		20		20
	Trend	↓		↓		↓		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	3.70		435.0		3.26		-0.10
	SD	0.25		74.0		0.22		0.10
	N	22		22		22		22
	Trend	-		> 0.05		.		.
Group: 3 o-xylene 100 mg/kg/day	Mean	3.60		412.1		3.19		-0.10
	SD	0.21		87.9		0.19		0.15
	N	21		21		21		21
	Trend	-		≤ 0.05	*	.		.
Group: 4 o-xylene 300 mg/kg/day	Mean	3.60		401.6		3.20		-0.15
	SD	0.21		77.1		0.24		0.16
	N	21		21		21		21
	Trend	> 0.05		≤ 0.05	*	> 0.05		> 0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05

Table 4 Food Intake (g/animal/day) Group Mean Values

Sex: Female		Mean Food Intake  (Day of Gestation)
Group		4 to 5 (#)		5 to 6 (#)		6 to 8 (#1)		8 to 10 (#1)		10 to 12 (#1)
Group: 1 Control 0 mg/kg/day	Mean	125.9		133.7		138.9		143.4		144.6
	SD	69.6		73.6		52.6		32.6		35.5
	N	21		21		21		20		20
	Trend	↓		↓		↓		↓		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	142.4		156.8		155.1		149.7		151.3
	SD	56.3		37.7		38.3		37.7		34.1
	N	22		22		22		22		22
	Trend	.		.		.		.		.
Group: 3 o-xylene 100 mg/kg/day	Mean	127.0		139.9		138.1		137.1		144.5
	SD	70.9		53.5		43.4		32.3		26.4
	N	21		21		20		20		20
	Trend	.		.		> 0.05		> 0.05		> 0.05
Group: 4 o-xylene 300 mg/kg/day	Mean	122.0		130.3		103.5		92.8		92.0
	SD	55.2		44.4		40.4		48.4		44.0
	N	21		21		20		20		20
	Trend	> 0.05		> 0.05		≤ 0.05	*	≤ 0.001	***	≤ 0.001	***

(#) - Shirley, Kruskal-Wallis & Steel

(#1) - Williams, Anova & Dunnett: * = p ≤ 0.05; *** = p ≤ 0.001

Sex: Female		Mean Food Intake  (Day of Gestation)
Group		12 to 14 (#)		14 to 16 (#)		16 to 18 (#)		18 to 20 (#)		20 to 22 (#)
Group: 1 Control 0 mg/kg/day	Mean	137.7		149.5		147.4		162.9		163.5
	SD	32.8		35.3		35.8		42.3		39.5
	N	20		20		19		20		20
	Trend	↓		↓		↓		↓		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	155.4		146.0		142.2		158.4		158.0
	SD	28.1		42.0		51.0		44.6		32.4
	N	22		22		22		22		21
	Trend	.		.		> 0.05		.		> 0.05
Group: 3 o-xylene 100 mg/kg/day	Mean	139.3		133.3		124.5		148.0		137.5
	SD	33.5		27.6		34.0		42.6		28.7
	N	20		20		21		21		20
	Trend	> 0.05		> 0.05		≤ 0.05	*	> 0.05		≤ 0.01	**
Group: 4 o-xylene 300 mg/kg/day	Mean	97.1		101.9		91.6		104.3		130.0
	SD	45.8		49.7		42.8		40.1		25.7
	N	19		19		17		17		16
	Trend	≤ 0.001	***	≤ 0.001	***	≤ 0.001	***	≤ 0.001	***	≤ 0.01	**

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

Sex: Female		Mean Food Intake  (Day of Gestation)						Overall Mean Food Intake (Day of Gestation)
Group		22 to 24 (#)		24 to 26 (#)		26 to 28 (#)		6 to 28 (#)
Group: 1 Control 0 mg/kg/day	Mean	126.3		88.1		88.1		135.7
	SD	34.4		32.2		34.3		24.7
	N	20		20		20		19
	Trend	↓		↓		↑		↓
Group: 2 o-xylene 30 mg/kg/day	Mean	119.1		85.2		95.2		140.0
	SD	30.0		26.2		31.7		19.0
	N	21		21		21		21
	Trend	.		.		.		.
Group: 3 o-xylene 100 mg/kg/day	Mean	98.6		70.2		81.7		123.5
	SD	39.1		31.8		32.2		23.8
	N	21		21		21		20
	Trend	.		.		.		> 0.05
Group: 4 o-xylene 300 mg/kg/day	Mean	115.3		93.3		99.7		107.7
	SD	25.1		36.4		29.9		23.9
	N	16		16		16		16
	Trend	> 0.05		> 0.05		> 0.05		≤ 0.001	***

(#) - Williams, Anova & Dunnett: *** = p ≤ 0.001

Table 5 Pregnancy Data Summary

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 o-xylene 30 mg/kg/day		Group: 3 o-xylene 100 mg/kg/day		Group: 4 o-xylene 300 mg/kg/day
Group Size		22		22		22		22
Not Pregnant		1		0		1		1
Not Pregnant %		4.5		0.0		4.5		4.5
Not Pregnant Died/Killed	Sum	0		0		0		0
Not Pregnant Schedule Kill	Sum	1		0		1		1
Pregnant		21		22		21		21
Pregnant %		95.5		100.0		95.5		95.5
Pregnant Died/Killed/Aborted	Sum	1		0		0		0
Pregnant with Total Resorption	Sum	0		0		0		0
Number with Live Foetuses	Sum	20		22		21		21

Table 6 Uterine and implantation data: Group mean values

Sex: Female		Group: 1	Group: 2	Group: 3	Group: 4
		Control	o-xylene	o-xylene	o-xylene
		0 mg/kg/day	30 mg/kg/day	100 mg/kg/day	300 mg/kg/day
Number with Live Foetuses (GD 28)		20	22	21	21
Number of Corpora Lutea (#)	Sum	217	237	205	204
	Mean	10.9	10.8	9.8	9.7
	SD	2.1	1.8	2	1.3
	Trend	↓	.	.	> 0.05
Number of Implantations (#1)	Sum	190	204	189	180
	Mean	9.5	9.3	9	8.6
	SD	2	1.9	2	2.1
	Trend	↓	.	.	> 0.05
% Pre-implantation Loss (#2)	Mean	11.9	13.3	7.7	11.5
	Trend	↓	.	.	> 0.05
Number of Early Deaths (#2)	Sum	2	6	3	6
	Mean	0.1	0.3	0.1	0.3
	SD	0.3	0.7	0.5	0.6
	Trend	↑	.	.	> 0.05

) - Williams, Anova & Dunnett(Log)

(#1) - Williams, Anova & Dunnett

(#2) - Shirley, Kruskal-Wallis & Steel

Table 6 Uterine and implantation data: Group mean values (continued)

Sex: Female		Group: 1		Group: 2		Group: 3		Group: 4
		Control		o-xylene		o-xylene		o-xylene
		0 mg/kg/day		30 mg/kg/day		100 mg/kg/day		300 mg/kg/day
Number of Late Deaths (#)	Sum	2		2		1		2
	Mean	0.1		0.1		0		0.1
	SD	0.3		0.3		0.2		0.3
	Trend	↓		.		.		> 0.05
Number of Dead Foetuses	Sum	0		0		0		0
	Mean	0		0		0		0
	SD	0		0		0		0
Number of Live Foetuses (#1)	Sum	186		196		185		172
	Mean	9.3		8.9		8.8		8.2
	SD	2		1.9		2.2		2.1
	Trend	↓		.		.		> 0.05
% Post-implantation Loss (#)	Mean	2.2		3.7		2.3		4
	Trend	↑		.		.		> 0.05
Live Foetuses as % of Implants	Mean	97.8		96.3		97.7		96

(#) - Shirley, Kruskal-Wallis & Steel

(#1) - Williams, Anova & Dunnett

Table 7 Litter weights (g)/ Foetal data - group mean values

Sex: Female		Group: 1		Group: 2		Group: 3		Group: 4
		Control		o-xylene		o-xylene		o-xylene
		0 mg/kg/day		30 mg/kg/day		100 mg/kg/day		300 mg/kg/day
Number with Live Foetuses		20		22		21		21
No of Live Foetuses		186		196		185		172
No of Male Foetuses		98		97		83		80
No of Female Foetuses		88		99		102		92
% of Male Foetuses (#)	Mean	52.8		48.4		45.1		45.5
	Trend	↓		.		.		> 0.05
Litter Weight (g) (#)	Mean	311.53		296.73		284.45		272.87
	Trend	↓		.		> 0.05		≤ 0.05	*
Foetal Weight (M+F) (g) (#)	Mean	33.88		33.7		32.57		33.88
	Trend	↓		.		.		> 0.05
Foetal Weight (M) (g)	Mean	34.18		34.81		32.73		34.24
Foetal Weight (F) (g)	Mean	33.61		32.62		32.29		33.53
Placental Weight (g) (#)	Mean	3.44		3.38		3.46		3.51
	Trend	↑		.		.		> 0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05

Data are recorded and reported against dams, day of lactation is equivalent to day of age 

Table 8 Examination of foetuses, summary of group mean values

Combined examination (external/visceral/ skeletal)
Dose level (mg/kg bw/ day)	0	30	100	300
	Group 1	Group 2	Group 3	Group 4
Total no. of litters examined	20	22	21	21
Total no. of foetuses examined	186	196	185	172
No. with major abnormalities	3	8	5	1
Mean % Foetuses Affected	1.8	4.0	2.3	0.4
No. of litters affected #	3	7	2	1
No. with minor abnormalities	104	100	81	79
Mean % Foetuses Affected	55.9	50.1	42.1	44.1
No. of litters affected #	20	21	21	18
No. with variations	186	194	183	169
Mean % Foetuses Affected	100.0	98.7	99.0	96.0
No. of litters affected #	20	22	21	21
# statistically analysed

Conclusions:

Administration of o-xylene to pregnant New Zealand White rabbits once daily by oral gavage, from Day 6 to Day 27 of gestation, inclusive, elicited maternal toxicity at 300 mg/kg/day in terms of transient post-dose observations of rapid breathing and pink/purple ears, and lower body weight gain and food intake. There was no adverse effect on pregnancy, embryonic or foetal development at any dose level. 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.

Executive summary:

The study objective was to investigate the effects of the test item, o-xylene, on the pregnant rabbit and developing organism when administered from Day 6 to Day 27 of gestation, inclusive. Four groups of 22 sexually mature, timed-mated, female New Zealand White rabbits were given o-xylene by gavage, at 0, (vehicle Control), 30, 100 or 300 mg/kg/day once daily at a dose volume of 5 mL/kg body weight from Day 6 to Day 27 of gestation, inclusive. All females were observed daily from the start of dosing and body weight and food intake were recorded at regular intervals. The animals were killed on Day 28 of gestation, a necropsy was performed and the internal organs were examined for macroscopic abnormalities. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral, skeletal (bone and cartilage) abnormalities and variants. Approximately 50 % of the foetuses in each litter were decapitated and fixed in Bouin’s fluid for subsequent fixed head examinations. One Control animal was killed on Day 15 of gestation following inappetence and body weight loss. Post-dose observations of pink/purple ears and rapid breathing were noted in most animals given 300 mg/kg/day throughout the dosing period. At 30 or 100 mg/kg/day, similar observations were noted in a few animals between Days 10 and 13 of gestation. At 300 mg/kg/day, overall mean body weight gain and food intake were significantly lower than Control. There was no adverse effect on body weight, body weight gain or food intake at 30 or 100 mg/kg/day o-xylene. There were 0, 22, 21 and 21 females with live foetuses on Day 28 of gestation in the groups given 0, 30, 100 or 300 mg/kg/day respectively. There was no effect on the pregnancy, uterine/implantation or foetal data. There was no effect of o-xylene administration on the nature, incidence or intergroup distribution of foetal abnormalities or variants. Administration of o-xylene to pregnant New Zealand White rabbits once daily by oral gavage, from Day 6 to Day 27 of gestation, inclusive, elicited maternal toxicity at 300 mg/kg/day in terms of transient post-dose observations of rapid breathing and pink/purple ears, and lower body weight gain and food intake. There was no adverse effect on pregnancy, embryonic or foetal development at any dose level. 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.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2019 to 16 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25th June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

The test item, p-xylene, was received on 21 June 2019 and on arrival it was given the Sequani log reference number TI/2019/096.
Details of the consignment received were:
Batch number SHBJ7329
Appearance Clear colourless liquid
Retest date (5 years after opening) 25 June 2024
Quantity supplied 6 x 1 litre
Purity 99.8 % (GC area %)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Specification
Eighty-eight timed-mated female New Zealand White rabbits were supplied by Envigo Global Services Inc. (formerly Covance Research Products Inc.), 310 Swampbridge Road, Denver, PA 17517, USA. Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation. The animals were approximately five months of age on arrival and on examination were found to be healthy. After at least three days acclimatisation, they were re-examined and confirmed to be suitable for use. On the first day of dosing the females weighed 2.65 kg to 3.8 kg.

Allocation and identification
Allocation to groups was performed using a stratified randomisation procedure based on individual body weights recorded on Day 0 of gestation at the suppliers (making sure that, where possible, females mated with the same male were spread across the groups). Each animal was uniquely identified by a subcutaneously implanted micro-identification device. To aid identification during procedures, where required, the animals were ear marked with their own unique identification numbers using an indelible pen. This mark was placed on the opposite ear to that of the supplier's number.

Housing and environmental conditions
The animals were housed individually in perforated-floor cages suspended over paper-lined trays. The study room was illuminated to give a cycle of 12 hours light and 12 hours dark and was air-conditioned. The target ranges for temperature and humidity were 16 °C to 20 °C and 40 % to 70 %, respectively. Recorded values were within, or marginally outside, these limits.

Diet and water
A pelleted diet, Teklad 2030C supplied by Envigo RMS (UK) Limited and mains tap water were freely available. From the day of arrival, approximately 30 g of hay was provided each day to animals as enrichment. Hay was supplied by IPS Product Supplies Ltd., BCM IPS Ltd., London, WC1N 3XX, United Kingdom. Where considered necessary, when food intake was reduced, feeding regimens were adjusted to encourage and maintain gut motility. Flavoured Bunny Blocks™ (supplied by LBS Servicing Biotechnology Ltd, PO Box 431, RH6 0UW, United Kingdom) were available and replaced each week.

Route of administration:

oral: gavage

Vehicle:

other: 1 % w/v methylcellulose (medium viscosity) with 1 % v/v Tween 80

Details on exposure:

Animals were dosed once daily from Day 6 to Day 27 of gestation inclusive, by gavage, using a rubber catheter and disposable syringe at a constant dose volume of 5 mL/kg body weight. Individual doses were adjusted according to the most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sets of samples (for analysis or for contingency) were taken from each test item formulation prepared throughout the study. All samples and reserve samples obtained from formulations on one occasion towards the start and end of the dosing period were sent to the Principal Investigator for analysis using validated method ST11FG (4). All remaining samples were retained and discarded once the final formulation analysis results were accepted.

Details on mating procedure:

Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.

Duration of treatment / exposure:

OECD 414 Main study. From Day 6 to Day 27 of gestation, inclusive.

Frequency of treatment:

Once daily

Duration of test:

From Day 6 to Day 28 of gestation

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Four groups of 22 sexually mature, timed-mated, female New Zealand White rabbits were given p-xylene by gavage, at 0 (vehicle Control), 30, 100 or 300 mg/kg/day once daily at a dose volume of 5 mL/kg body weight from Day 6 to Day 27 of gestation, inclusive. All females were observed daily from the start of dosing and body weight and food intake were recorded at regular intervals. The animals were killed on Day 28 of gestation, a necropsy performed and the internal organs were examined for macroscopic abnormalities. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral and skeletal (bone and cartilage) abnormalities and variants. Approximately 50 % of the foetuses in each litter were decapitated and fixed in Bouin’s fluid for subsequent fixed head examination.

Maternal examinations:

Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily. From the start of dosing, animals were observed before and shortly after dosing. On weekdays, animals were also observed at one hour after dosing and at the weekend they were observed either at one hour after dosing or at the end of the working day.

Body weights were recorded on Day 0 of gestation by the supplier. At Sequani, body weights were recorded daily from Day 4 to Day 28 of gestation, inclusive.

The amount of food consumed by each animal was recorded daily over Days 4 to 6 of gestation and every two days thereafter.

The females were killed on Day 28 of gestation by an overdose injection of sodium pentobarbitone. The animals were weighed, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined.

Ovaries and uterine content:

Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained in 70 % IDA (industrial denatured alcohol) for approximately seven days and then transferred and retained in 10 % buffered formalin.

For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix. The foetuses and their placentae were removed and the uterus and ovaries retained in 10 % buffered formalin.

Fetal examinations:

On Day 28 of gestation, live foetuses were killed by an intraperitoneal injection of sodium pentobarbitone solution, weighed and examined for external abnormalities. The foetuses were placed in 70 % IDA. Later in the day, the viscera were examined and the sex recorded. For 50 % of the foetuses, they were eviscerated and a coronal section was made through the head along the frontal parietal suture and the brain examined. For the remaining 50 % of foetuses, they were eviscerated and decapitated and the heads placed in Bouin’s fluid for subsequent serial sectioning. The foetuses (intact and decapitated) were then placed in a water bath and then skinned to prepare the carcasses for staining. The carcasses were cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined.

Structural congenital abnormalities that impair, or potentially impair, the survival or constitution of the foetus were classified as major abnormalities. Abnormalities that in isolation, do not impair the survival or constitution of the foetus but could potentially interfere with normal bodily function were classified as minor abnormalities. Alternative structures occurring regularly in the control population, which may be permanent or transient, were classified as variants. Foetuses with major external or visceral abnormalities were photographed. For archiving, all foetal heads fixed in Bouin's fluid were stored in 10 % buffered formalin and all skeletal specimens were stored in aqueous glycerol with thymol crystals (to prevent fungal growth)

Statistics:

Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1. Depending on the nature of the data set that was to be analysed, appropriate tests were applied, as indicated in the table below. Where parametric tests were appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the ShapiroWilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied. Proportions of foetuses affected were treated as continuous non-parametric data, using onesided step-wise Jonckheere Tests. Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.

Indices:

Pre-implantation loss (%) = (no. of corpora lutea – no. of implantation sites)/ no. of corpora lutea x 100
Post-implantation loss (%) = (no. of implantation sites – no. of live foetuses)/ no. of implantation sites x 100
Mean pre- and post-implantation losses were calculated on a proportional litter basis. Mean foetal body weights were calculated separately by sex for each litter; group mean body weights were calculated (separately by sex) from the litter means. The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.

Historical control data:

Historical control data available see attached 'background material'.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, pink/purple ears were observed in 15 animals throughout the dosing period and seven females showed rapid breathing for up to 10 days. These observations were seen immediately after dosing but were no longer present at the one hour observation time-point. One female given 30 mg/kg/day, showed subdued and unsteady gait immediately after dosing on Days 22 and 23 of gestation.
There were no clinical observations at 100 mg/kg/day following administration of p-xylene.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, mean overall body weight gain was slightly, but not statistically significantly, lower than Controls. There were no effects on body weight or body weight gain at 30 or 100 mg/kg/day. Terminal body weight adjusted for the weight of the gravid uterus was similar in all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was statistically significantly (p ≤ 0.01) lower than Controls over the dosing period for females given 300 mg/kg/day. One female given 30 mg/kg/day, and one female given 300 mg/kg/day, were offered moistened diet and/or carrots to encourage eating following a decline in food intake during the dosing period. In addition, one female given 100 mg/kg/day and one given 300 mg/kg/day, did not eat from arrival and were also offered moistened diet and/or carrots.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Three Control females, one in each of the groups given 30 or 100 mg/kg/day and four females given 300 mg/kg/day were not pregnant. This resulted in 19, 21, 21 and 18 females with live foetuses on Day 28 of gestation, in the groups given 0, 30, 100 or 300 mg/kg/day, respectively. There was no effect on the number of implantations, incidences of pre- or post-implantation loss or on the number of live foetuses that were attributed to p-xylene.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, mean foetal weight was statistically significantly lower than Controls (p ≤ 0.01) and slightly below the historical Control data range; however, the lower foetal weights were considered to be a consequence of the reduction in maternal body weight gain in the group, and not an adverse effect of p-xylene administration. There was no effect of p-xylene on mean foetal weight at 30 or 100 mg/kg/day and no effect on mean placental weight or on the foetal sex ratio at any dose level.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect on the number of live foetuses that were attributed to p-xylene.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of p-xylene on the foetal sex ratio at any dose level.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of some minor and variant abnormalities were higher in the groups administered p-xylene, such as 27 pre-sacral vertebrae (minor), incomplete or misshapen cartilaginous plate of the cervical vertebra (variant) and other minor and variant abnormalities relating to incomplete or non-ossification of some small bones. Incomplete or non-ossification of small bones is related to the smaller foetal sizes and as either the litter and/or foetal incidences were within, or only marginally above, the historical Control data ranges, these changes were considered not to be an adverse effect of p-xylene administration. The nature, incidence and inter-group distribution of external, visceral and skeletal foetal abnormalities and variations did not indicate and adverse effect of p-xylene administration.

Visceral malformations:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Developmental effects observed:

no

Treatment related:

no

Table 1 Body weights (kg) - Group Mean Values

Sex: Female		Body Weight (Day of Gestation)
Group		0		4		5		6 (#)		7		8
Group: 1 Control 0 mg/kg/day	Mean	2.968		2.913		2.981		3.013		3.032		3.041
	SD	0.176		0.188		0.202		0.197		0.202		0.203
	N	19		19		19		19		19		19
	Trend	.		.		.		↑		.		.
Group: 2 p-xylene 30 mg/kg/day	Mean	3.006		2.969		3.030		3.058		3.066		3.063
	SD	0.232		0.240		0.253		0.252		0.249		0.243
	N	21		21		21		21		21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	2.993		2.951		2.994		3.025		3.066		3.063
	SD	0.210		0.215		0.223		0.216		0.226		0.224
	N	21		21		21		21		21		21
Group: 4 p-xylene 300 mg/kg/day	Mean	3.026		2.965		3.035		3.051		3.053		3.050
	SD	0.263		0.254		0.251		0.264		0.277		0.279
	N	18		18		18		18		18		18
	Trend	.		.		.		> 0.05		.		.

(#) - Shirley, Kruskal-Wallis & Steel

Sex: Female		Body Weight (Day of Gestation)
Group		9		10		11		12		13		14
Group: 1 Control 0 mg/kg/day	Mean	3.048		3.055		3.069		3.091		3.118		3.144
	SD	0.204		0.209		0.215		0.210		0.207		0.211
	N	19		19		19		19		19		19
	Trend	.		.		.		.		.		.
Group: 2 p-xylene 30 mg/kg/day	Mean	3.075		3.088		3.106		3.129		3.161		3.186
	SD	0.246		0.242		0.237		0.244		0.246		0.242
	N	21		21		21		21		21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	3.084		3.095		3.107		3.120		3.138		3.169
	SD	0.231		0.227		0.218		0.212		0.211		0.214
	N	21		21		21		21		21		21
Group: 4 p-xylene 300 mg/kg/day	Mean	3.053		3.072		3.093		3.118		3.135		3.159
	SD	0.272		0.272		0.268		0.265		0.261		0.250
	N	18		18		18		18		18		18
	Trend	.		.		.		.		.		.

 

Sex: Female		Body Weight (Day of Gestation)
Group		21		22		23		24		25		26
Group: 1 Control 0 mg/kg/day	Mean	3.303		3.330		3.336		3.342		3.366		3.385
	SD	0.228		0.232		0.239		0.245		0.234		0.234
	N	19		19		19		19		19		19
	Trend	.		.		.		.		.		.
Group: 2 p-xylene 30 mg/kg/day	Mean	3.339		3.346		3.348		3.353		3.378		3.397
	SD	0.256		0.258		0.250		0.247		0.248		0.240
	N	21		21		21		21		21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	3.308		3.322		3.340		3.350		3.361		3.381
	SD	0.240		0.235		0.234		0.246		0.244		0.246
	N	21		21		21		21		21		21
Group: 4 p-xylene 300 mg/kg/day	Mean	3.298		3.311		3.325		3.332		3.339		3.353
	SD	0.247		0.255		0.246		0.246		0.242		0.247
	N	18		18		18		18		18		18
	Trend	.		.		.		.		.		.

 

Sex: Female		Body Weight (Day of Gestation)
Group		27		28 (#)
Group: 1 Control 0 mg/kg/day	Mean	3.394		3.422
	SD	0.236		0.238
	N	19		19
	Trend	.		↓
Group: 2 p-xylene 30 mg/kg/day	Mean	3.410		3.434
	SD	0.234		0.235
	N	21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	3.385		3.419
	SD	0.244		0.235
	N	21		21
Group: 4 p-xylene 300 mg/kg/day	Mean	3.371		3.400
	SD	0.252		0.248
	N	18		18
	Trend	.		> 0.05

(#) - Shirley, Kruskal-Wallis & Steel

Table 2 Body weight gains (kg) - group mean values

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		5 to 6 (#)		0 to 6 (#)		6 to 7 (#)		6 to 8 (#)		6 to 9 (#)		6 to 12 (#)		6 to 15 (#1)
Group: 1 Control 0 mg/kg/day	Mean	0.032		0.044		0.019	*	0.028		0.035		0.078		0.167
	SD	0.049		0.098		0.023		0.035		0.038		0.050		0.054
	N	19		19		19		19		19		19		19
	Trend	↓		↓		↓		↓		↓		↓		↓
Group: 2 p-xylene 30 mg/kg/day	Mean	0.027		0.052		0.009		0.005		0.018		0.071		0.167
	SD	0.051		0.103		0.035		0.041		0.046		0.058		0.077
	N	21		21		21		21		21		21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	0.031		0.032		0.040		0.038		0.059		0.095		0.180
	SD	0.048		0.242		0.055		0.067		0.077		0.080		0.071
	N	21		21		21		21		21		21		21
	Trend	.		.		.		> 0.05		> 0.05		.		.
Group: 4 p-xylene 300 mg/kg/day	Mean	0.016		0.025		0.002		-0.001		0.002		0.067		0.134
	SD	0.046		0.107		0.038		0.042		0.045		0.074		0.086
	N	18		18		18		18		18		18		18
	Trend	> 0.05		> 0.05		> 0.05		≤ 0.05	*	≤ 0.05	*	> 0.05		> 0.05

(#) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05

(#1) - Williams, Anova & Dunnett

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		5 to 6 (#)		0 to 6 (#)		6 to 7 (#)		6 to 8 (#)		6 to 9 (#)		6 to 12 (#)		6 to 15 (#1)
Group: 1 Control 0 mg/kg/day	Mean	0.032		0.044		0.019	*	0.028		0.035		0.078		0.167
	SD	0.049		0.098		0.023		0.035		0.038		0.050		0.054
	N	19		19		19		19		19		19		19
	Trend	↓		↓		↓		↓		↓		↓		↓
Group: 2 p-xylene 30 mg/kg/day	Mean	0.027		0.052		0.009		0.005		0.018		0.071		0.167
	SD	0.051		0.103		0.035		0.041		0.046		0.058		0.077
	N	21		21		21		21		21		21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	0.031		0.032		0.040		0.038		0.059		0.095		0.180
	SD	0.048		0.242		0.055		0.067		0.077		0.080		0.071
	N	21		21		21		21		21		21		21
	Trend	.		.		.		> 0.05		> 0.05		.		.
Group: 4 p-xylene 300 mg/kg/day	Mean	0.016		0.025		0.002		-0.001		0.002		0.067		0.134
	SD	0.046		0.107		0.038		0.042		0.045		0.074		0.086
	N	18		18		18		18		18		18		18
	Trend	> 0.05		> 0.05		> 0.05		≤ 0.05	*	≤ 0.05	*	> 0.05		> 0.05

(#) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05

(#1) - Williams, Anova & Dunnett

 

Sex: Female		Body Weight Gain  (Day of Gestation)
Group		6 to 19 (#)		6 to 22 (#)		6 to 25 (#)		6 to 28 (#)
Group: 1 Control 0 mg/kg/day	Mean	0.243		0.317		0.354		0.409
	SD	0.060		0.077		0.079		0.079
	N	19		19		19		19
	Trend	↓		↓		↓		↓
Group: 2 p-xylene 30 mg/kg/day	Mean	0.236		0.288		0.320		0.376
	SD	0.094		0.114		0.122		0.130
	N	21		21		21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	0.243		0.297		0.336		0.394
	SD	0.112		0.111		0.127		0.121
	N	21		21		21		21
	Trend	.		.		.		.
Group: 4 p-xylene 300 mg/kg/day	Mean	0.205		0.261		0.288		0.349
	SD	0.093		0.102		0.113		0.120
	N	18		18		18		18
	Trend	> 0.05		> 0.05		> 0.05		> 0.05

(#) - Williams, Anova & Dunnett

Table 3 Terminal Body Weights adjusted for gravis uterus weight - group mean values

Sex: Female		Dead Body Weight  (kg)		Gravid Uterus Weight (g)		Body Weight Adjusted for Gravid  Uterus Weight (kg)		Adjusted Body Weight Gain (kg) (Day of Gestation)
Group		(#)		(#)		(#)		6 to 28 (#)
Group: 1 Control 0 mg/kg/day	Mean	3.42		428.2		2.99		-0.03
	SD	0.25		83.4		0.24		0.09
	N	19		19		19		19
	Trend	↓		↓		↑		↓
Group: 2 p-xylene 30 mg/kg/day	Mean	3.43		440.9		3.05		-0.11
	SD	0.24		82.8		0.23		0.14
	N	21		21		21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	3.41		399.1		3.03		-0.03
	SD	0.23		45.2		0.23		0.15
	N	21		21		21		21
Group: 4 p-xylene 300 mg/kg/day	Mean	3.39		398.3		3.00		-0.09
	SD	0.25		54.5		0.24		0.13
	N	18		18		18		18
	Trend	> 0.05		> 0.05		> 0.05		> 0.05

Table 4: Food intake (g/animal/day) - group mean values

Sex: Female		Mean Food Intake  (Day of Gestation)
Group		4 to 5 (#)		5 to 6 (#)		6 to 8 (#)		8 to 10 (#1)		10 to 12 (#1)		12 to 14 (#1)
Group: 1 Control 0 mg/kg/day	Mean	159.1		160.2		160.3		149.1		147.6		140.7
	SD	57.0		43.0		27.5		30.2		31.7		24.9
	N	19		19		18		19		19		19
	Trend	↓		↓		↓		↓		↓		↓
Group: 2 p-xylene 30 mg/kg/day	Mean	160.9		162.4		148.0		150.0		150.4		143.5
	SD	58.3		22.9		23.0		26.4		26.4		27.8
	N	21		21		21		21		21		21
Group: 3 p-xylene 100 mg/kg/day	Mean	143.0		152.8		154.5		154.0		143.5		140.0
	SD	66.6		41.7		30.5		31.6		29.7		36.7
	N	21		21		21		21		21		21
	Trend	.		.		> 0.05		> 0.05		> 0.05		> 0.05
Group: 4 p-xylene 300 mg/kg/day	Mean	158.6		149.0		125.1		126.7		128.4		119.3
	SD	55.8		43.0		37.5		26.7		20.0		24.6
	N	18		18		18		17		17		17
	Trend	> 0.05		> 0.05		≤ 0.001	***	≤ 0.05	*	≤ 0.05	*	≤ 0.05	*

(#) - Shirley, Kruskal-Wallis & Steel: *** = p ≤ 0.001

(#1) - Williams, Anova & Dunnett: * = p ≤ 0.05

Sex: Female		Mean Food Intake  (Day of Gestation)
Group		14 to 16 (#)		16 to 18 (#)		18 to 20 (#)		20 to 22 (#1)		22 to 24 (#)		24 to 26 (#)
Group: 1 Control 0 mg/kg/day	Mean	151.7		145.3		163.9		153.0		120.5		105.2
	SD	34.4		32.6		25.1		27.1		26.2		26.2
	N	19		19		19		18		19		18
	Trend	↓		↓		↓		↓		↓		↓
Group: 2 p-xylene 30 mg/kg/day	Mean	145.3		149.6		167.5		143.5		120.1		112.2
	SD	36.8		38.2		29.1		38.9		23.5		26.3
	N	21		21		21		21		20		20
Group: 3 p-xylene 100 mg/kg/day	Mean	143.5		142.3		156.7		146.7		125.6		117.4
	SD	35.9		45.5		47.2		36.3		34.2		37.9
	N	21		21		21		21		21		21
	Trend	> 0.05		.		.		> 0.05		.		.
Group: 4 p-xylene 300 mg/kg/day	Mean	115.9		121.4		142.6		124.9		104.4		97.6
	SD	28.1		41.5		25.9		26.6		24.3		27.9
	N	17		18		18		18		18		18
	Trend	≤ 0.01	**	> 0.05		> 0.05		≤ 0.05	*	> 0.05		> 0.05

(#) - Williams, Anova & Dunnett: ** = p ≤ 0.01

(#1) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05

Sex: Female		Mean Food Intake  (Day of Gestation)		Overall Mean Food Intake (Day of Gestation)
Group		26 to 28 (#)		6 to 28 (#1)
Group: 1 Control 0 mg/kg/day	Mean	108.2		140.1
	SD	25.3		20.6
	N	19		17
	Trend	↓		↓
Group: 2 p-xylene 30 mg/kg/day	Mean	113.9		140.6
	SD	19.8		15.2
	N	20		20
Group: 3 p-xylene 100 mg/kg/day	Mean	116.0		140.0
	SD	33.7		30.0
	N	21		21
	Trend	.		> 0.05
Group: 4 p-xylene 300 mg/kg/day	Mean	102.6		118.7
	SD	21.0		17.1
	N	18		17
	Trend	> 0.05		≤ 0.01	**

(#) - Williams, Anova & Dunnett(Log)

(#1) - Williams, Anova & Dunnett: ** = p ≤ 0.01

Table 5: Pregnancy data - summary

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 p-xylene 30 mg/kg/day		Group: 3 p-xylene 100 mg/kg/day		Group: 4 p-xylene 300 mg/kg/day
Group Size		22		22		22		22
Not Pregnant		3		1		1		4
Not Pregnant %		13.6		4.5		4.5		18.2
Not Pregnant Died/Killed	Sum	0		0		0		0
Not Pregnant Schedule Kill	Sum	3		1		1		4
Pregnant		19		21		21		18
Pregnant %		86.4		95.5		95.5		81.8
Pregnant Died/Killed/Aborted	Sum	0		0		0		0
Pregnant with Total Resorption	Sum	0		0		0		0
Number with Live Foetuses	Sum	19		21		21		18

Table 6: Uterine and implantation data - group mean values

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 p-xylene 30 mg/kg/day		Group: 3 p-xylene 100 mg/kg/day		Group: 4 p-xylene 300 mg/kg/day
Number with Live Foetuses (GD 28)		19		21		21		18
Number of Corpora Lutea (#)	Sum	183		188		195		167
	Mean	9.6		9.0		9.30		9.3
	SD	1.6		1.4		1.3		1.4
	Trend	↓		.		.		> 0.05
Number of Implantations (#1)	Sum	166		171		176		157
	Mean	8.7		8.1		8.4		8.7
	SD	2.1		1.1		1.7		1.4
	Trend	↓		.		.		> 0.05
% Pre-implantation Loss (#2)	Mean	9.7		7.3		9.4		6.0
	Trend	↓		.		.		> 0.05
Number of Early Deaths (#2)	Sum	2*		1		15		2
	Mean	0.1*		0.0		0.7		0.1
	SD	0.3		0.2		1.4		0.3
	Trend	↑		.		.		> 0.05

(#) - Williams, Anova & Dunnett

(1#) - Shirley, Kruskal-Wallis & Steel: *= p≤0.05

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 p-xylene 30 mg/kg/day		Group: 3 p-xylene 100 mg/kg/day		Group: 4 p-xylene 300 mg/kg/day
Number of Late Deaths (#)	Sum	2		2		1		1
	Mean	0.1		0.1		0.0		0.1
	SD	0.3		0.3		0.2		0.2
	Trend	↓		.		.		> 0.05
Number of Dead Foetuses	Sum	0		0		0		0
	Mean	0.0		0.0		0.0		0.0
	SD	0.0		0.0		0.0		0.0
Number of Live Foetuses (#1)	Sum	162		168		160		154
	Mean	8.5		8.0		7.6		8.6
	SD	2.1		1.1		2.2		1.4
	Trend	↓		.		.		> 0.05
% Post-implantation Loss (#)	Mean	2.4		1.7		9.3		1.7
	Trend	↑		.		.		> 0.05
Live Foetuses as % of Implants	Mean	97.6		98.3		90.7		98.3

(#) - Williams, Anova & Dunnett

(1#) - Shirley, Kruskal-Wallis & Steel: *= p≤0.05

Table 7: Litter weights (g) / foetal data - group mean values

Sex: Female		Group: 1 Control 0 mg/kg/day		Group: 2 p-xylene 30 mg/kg/day		Group: 3 p-xylene 100 mg/kg/day		Group: 4 p-xylene 300 mg/kg/day
Number with Live Foetuses		19		21		21		21
No of Live Foetuses		162		168		160		154
No of Male Foetuses		83		85		83		65
No of Female Foetuses		79		83		77		89
% of Male Foetuses (#)	Mean	49.8		51.6		51.4		42.6
	Trend	↓		.		.		> 0.05
Litter Weight (g) (#)	Mean	295.58		275.04		265.67		271.18
	Trend	↓		.		.		> 0.05
Foetal Weight (M+F) (g) (#)	Mean	35.13		34.62		35.36		31.96
	Trend	↓		.		>0.05		> 0.05
Foetal Weight (M) (g)	Mean	35.6		34.78		36.21		32.97
Foetal Weight (F) (g)	Mean	34.48		34.57		34.30		31.06
Placental Weight (g) (#)	Mean	3.53		3.38		3.76		3.31
	Trend	↓		.		.		> 0.05

(#) - Williams, Anova & Dunnett: * = p≤0.05; ** = p≤0.01

Table 8: Examination of foetuses - summary of group mean values

	Control	Group 2	Group 3	Group 4
Dose Level mg/kg bw/day :	0	30	100	300
Total Number of Litters Examined:	19	21	21	18
Total Number of Foetuses Examined:	162	168	160	154
Number with Major Abnormalities:	0	1	2	2
Mean % of Foetuses Examined	0.0	0.6	1.0	1.3
Number of Litters Affected#	0	1	2	2
Number with Minor Abnormalities:	73	66	91	97
Mean % of Foetuses Examined	44.1	37.8	54.8	62.5JJ
Number of Litters Affected#	19	18	19	18
Number with Variations:	161	167	160	154
Mean % of Foetuses Examined	99.5	99.4	100.0	100.0
Number of Litters Affected#	19	21	21	18

# Statistically Analysed

JJ = p<0.01 (Jonckheere Test)

Table 9: Examination of foetuses - group mean values-by foetus

Key finding	Type	Control	Group 2	Group 3	Group 4
Thoracic cavity (total number of foetuses examined (total number of litters examined))		162 (19)	168 (21)	160 (21)	154 (18)
subclavian artery- right:  retro-oesophageal	minor	0(0.0)	0(0.0)	0(0.0)	2J(1.4)
Skull (total number of foetuses examined (total number of litters examined))		82 (19)	88 (21)	77 (21)	77 (18)
Parietal- uni- or bilateral: incomplete ossification	minor	0(0.0)	0(0.0)	1(1.0)	3J(3.4)
parietal- uni- or bilateral:  incomplete ossification	minor	0(0.0)	0(0.0)	1)4.8)	3C(16.7)
Vertebra (total number of foetuses examined (total number of litters examined))		162 (19)	168 (21)	160 (21)	154 (18)
number of presacral vertebra: 27	minor	14(8.4)	14(8.7)	23 (17.4)	30J(20.9)
Cervical vertebra
cartilaginous ventral plate- uni- or bilateral: incomplete	variant	20(12.4)	19(11.1)	41J(24.4)	28J(18.4)
Thoracic vertebra
1 or more centra: ventral crest present	variant	0(0.0)	1(0.7)	0(0.0)	4J(2.8)
Caudal vertebra (see above)	variant	10(4.8)	10(5.6)	17(10.2)	26JJJ(16.5)
Sternum
5th sternebra: incomplete ossification	variant	53(32.5)	64(37.6)	77J(49.1)	74JJ(48.4)
Pelvic girdle
pubis- uni- or bilateral: not ossified	minor	1(0.6)	1(0.5)	4(2.7)	12J(7.3)
Forelimb
epiphyses- uni- or bilateral: not ossified	variant	25(13.6)	18(10.1)	46J(29.7)	44J(27.4)
1 or more digit: phalanges not ossified	minor	0(0.0)	1(0.6)	4J(2.3)	5J(3.2)
1 or more metacarpal: not ossified	minor	29(17.4)	17(9.5)	45(26.3)	56J(35.2)
Hindlimb
epiphyses- uni- or bilateral: not ossified	variant	119(70.9)	124(72.9)	137J(85.7)	125J(80.9)
1 or more digit: phalanges incomplete ossification	variant	1(0.8)	2(1.3)	12JJ(7.0)	24JJJ(15.0)

Number of foetuses affected (group mean percent)

J = p<0.05; JJ = p<0.05; JJJ = p<0.001 (Jonckheere Test)

Conclusions:

Administration of p-xylene to pregnant New Zealand White rabbits once daily by oral gavage, from Day 6 to Day 27 of gestation, inclusive, elicited slight maternal toxicity at 300 mg/kg/day in terms of transient post-dose observations of rapid breathing and pink/purple ears, and lower body weight gain and food intake resulting in lower foetal weights. There was no adverse effect on pregnancy or on embryonic or foetal development at any dose level. 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.

Executive summary:

The objective of this study was to investigate the effects of the test item, p-xylene, on the pregnant New Zealand White rabbit and developing organism when administered from Day 6 to Day 27 of gestation, inclusive. Four groups of 22 sexually mature, timed-mated, female New Zealand White rabbits were given p-xylene by gavage, at 0 (vehicle Control), 30, 100 or 300 mg/kg/day once daily at a dose volume of 5 mL/kg body weight from Day 6 to Day 27 of gestation, inclusive. All females were observed daily from the start of dosing and body weight and food intake were recorded at regular intervals. The animals were killed on Day 28 of gestation, a necropsy performed and the internal organs were examined for macroscopic abnormalities. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral and skeletal (bone and cartilage) abnormalities and variants. Approximately 50 % of the foetuses in each litter were decapitated and fixed in Bouin’s fluid for subsequent fixed head examination. There were no deaths during the study. Post-dose observations of pink/purple ears were noted in 15 animals given 300 mg/kg/day throughout the dosing period and six females showed rapid breathing for up to 10 days, immediately after dosing. One female given 30 mg/kg/day showed subdued and unsteady gait immediately after dosing on Days 22 and 23 of gestation. There were no clinical observations at 100 mg/kg/day. At 300 mg/kg/day, overall mean body weight gain was slightly lower than Controls and overall mean food intake was statistically significantly lower than Controls. Terminal body weight adjusted for the weight of the gravid uterus, was similar in all groups. There was no adverse effect on body weight, body weight gain or food intake at 30 or 100 mg/kg/day p-xylene. There was no effect on the pregnancy or uterine /implantation data. At 300 mg/kg/day, mean foetal weight was statistically significantly lower than Controls, and slightly lower than the historical Control data range. There was no effect on placental weight or on the foetal sex ratio. There was no effect of p-xylene administration on the nature, incidence or intergroup distribution of foetal abnormalities or variations. Administration of p-xylene to pregnant New Zealand White rabbits once daily by oral gavage, from Day 6 to Day 27 of gestation, inclusive, elicited maternal toxicity at 300 mg/kg/day in terms of transient post-dose observations of rapid breathing and pink/purple ears, and lower body weight gain and food intake resulting in lower foetal weights. There was no adverse effect on pregnancy or on embryonic or foetal development at any dose level. 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.

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.