Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status not known, non-guideline animal experimental study, limitations in design and/or reporting but otherwise adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
no guideline followed
Deviations:
not applicable
Principles of method if other than guideline:
One generation reproduction study in CD rats.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): mixed xylenes (technical grade)
- Physical state: clear liquid
- Analytical purity: 87-92% ai
- Composition of test material, percentage of components:
2.4% toluene
12.8% ethylbenzene
20.3% para-xylene
44.2% meta-xylene
20.4% ortho-xylene
Source: American Petroleum Institute
- Lot/batch No.: 81-01 (Batch No. 1), UN 1307 RQ
- Stability: Fully analysed prior to study initiation. Additionally, each container (55 gallon drum) of mixed xylenes was sampled for analysis at the start of use and once during use.
- Expiration date: May 1983
- Storage condition of test material: In a ventilated, temperature controlled area (<75°F).

Test animals

Species:
rat
Strain:
other: Crl-CD® (SC) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., St. Constance, Ontario, Canada
- Age at study initiation: 50 days
- Weight at study initiation: group mean range: 223-226 g (males), 162-165 g (females)
- Fasting period before study: none
- Housing: During the study, animals were caged individually in stainless steel wire mesh cages, with the exception of mating (nightly co-housing of males with females) and during lactation (female with the litter during non-exposure intervals)
- Diet: Purina Laboratory Chow 5002 (mash) ad libitum except during exposure periods
- Water: tap water ad libitum
- Acclimation period: 20 days (June 3-23, 1981)
- On day 21 of gestation, each female's cage was fitted with a stainless steel floor pan; these were removed on day 14 of lactation.
- Litter Kleen® hardwood shavings were added to the females cages on day 21 of gestation and fresh bedding provided as necessary. Bedding was removed on day 14 of lactation.

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

IN-LIFE DATES: From: 24 June 1981 To: 4 January 1982

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.