Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 29, 1996 to May 15, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study that was performed according to OECD Guideline 414.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,6-dimethylphenol
- Physical state: white solid at ambient storage with sweet, tarry odor
- Analytical purity: Considered 100 % for purposes of preparing dosing solutions.
- Storage condition of test material: ambient temperature in a closed container.

Test animals

Species:
rat
Strain:
other: CD-Crl: [SD] BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 64 days (at initiation of mating)
- Weight at study initiation: 195.1-255.4 g (Gestation Day 0)
- Housing: Individually, except during mating, in stainless steel suspended cages with wire mesh floors and fronts.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 46-72
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1996-07-23 To: 1996-08-23

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of test substance were mixed with the corn oil to provide concentration levels of 12, 36, and 108 mg/mL for the low, mid and high dose groups, respectively. In addition, an appropriate amount of vehicle was dispensed for the control animals. Fresh dosing solutions were prepared at each concentration level weekly and dispensed for dosing daily. All dosing solutions were stored at ambient temperature. 5 mL/kg of body weight/day was administered to the animals. Initial dose volumes administered to individual animals were derived from Day 6 gestation body weights. The dose volumes were adjusted on days 9 and 12 of gestation to changes in individual animal body weights. Animals were dosed at approximately the same time each day.

VEHICLE
- Concentration in vehicle: 12, 36 and 108 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): 125H0892, 56H0124
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initiation of the study, mock batches of dosing solutions at the low and high levels were prepared. To assess homogeneity, three samples were collected from each of the top, middle and bottom portions of the containers with these formulations after mixing and analyzed. Formulations were considered homogeneous if the three samples at each level analyzed were within +/-10 % of each other and of the nominal concentration level. Stability of the test substance in the vehicle under ambient storage was established over a 14-day period at concentrations that ranged from 14 to 200 mg/mL. These analyses were performed in a pilot developmental toxicity study conducted by the laboratory. Since the concentration level for this study (i.e. 12 mg/ml) was outside this range, additional stability analyses were performed at this concentration only. Duplicate samples of the mock batch of solution prepared to assess homogeneity were assayed at 7 and 14 days post-preparation (Day 0 homogeneity assays, were used to establish the concentration at time of preparation).Dosing formulations for all three dose levels from the first three mixes used in study were assayed prior to use in the study (one sample per concentration was taken and analyzed in duplicate). The formulations were considered acceptable for use in study if the two samples from each concentration level were within 10% of each other.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1; The evenings for cohabitation of males with females were scheduled to provide female at Day 20 gestation sacrifice during the Monday-to-Friday work week.
- Length of cohabitation: Mating was conducted on 10 nights as follows: 23-27, 30 and 31 July and 1-3 August 1996.
- Proof of pregnancy: Vaginal smears were taken early in the morning following intervals of nightly cohabitation. Females were considered to have mated if sperm was noted microscopically in the vaginal rinse and/or a plug was observed in the vaginal opening. The day on which this evidence of mating was observed was defined as day 0 of gestation.
Duration of treatment / exposure:
Animals were treated once daily by oral gavage.
Frequency of treatment:
Animals were treated daily during the day 6-15 gestation interval.
Duration of test:
Animals were sacrificed on Day 20 of gestation.
Doses / concentrations
Remarks:
Doses / Concentrations:
60, 180, and 540 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): Females which mated were assigned to the groups daily in such a way as to provide an equal distribution of females among groups and equalize, as best possible, the Day 0 gestation mean body weights between groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: animals were observed for signs of pharmacologic or toxicologic effect and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 0, 6-16, 18 and 20 of gestation; On days 6-15 of gestation, a detailed physical examination was conducted approximately one half hour after animals had been dosed.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 12, 16, 18, and 20 of gestation; Day 20 gestation body weights were presented as actual and corrected (actual body weight minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined: Yes; Days 0-6, 6-9, 9-12, 12-16 and 16-20 (results reported as g/kg/day)


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Complete macroscopic postmortem examinations were performed on all test animals. External surfaces, all orifices, the cranial cavity, carcass, the external surface of the spinal cord and sectioned surfaces of the brain, nasal cavity and paranasal sinuses, the thoracic, abdominal and pelvic cavities and their viscera and the cervical tissues and organs were examined. Gross lesions noted during the postmortem evaluations were retained and preserved in 10% neutral buffered formalin. The carcass of each female was discarded at completion of the postmortem evaluation. The livers were weighed for all females and liver/body weight ratios were calculated using the corrected Day 20 question weights.

Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes; The intact uterus (ovaries attached) was removed from the abdominal cavity. The ovaries were dissected from the uterus and evaluated. When no uterine implants were grossly apparent, the uterus was stained with ammonium sulfide to indentify foci of implantation and early foetal death. When no foci were visualized post staining, the female was considered not pregnant.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes (total of fetuses plus resorptions)
- Number of early resorptions: Yes (evidence of implantation but no recognizable fetus)
- Number of late resorptions: Yes (recognizable dead fetus undergoing degeneration, regardless of size)
- Other: The number of live (movement to touch) and dead fetuses (lack of movement in response to touch with no visible degeneration) were measured.
Fetal examinations:
- External examinations: Yes: all per litter; Each fetus was individually identified, weighed, sexed externally (ano-genital distance) and given a gross examination for external malformation/variations to include observation for palatel defects.
- Soft tissue examinations: Yes: half per litter; The selected fetuses in each litter (alternating fetuses within the litter) were evaluated for malformation/variations using a microdissection procedure. Evaluations were performed on the fresh fetal specimens shortly after removal from the uterus. Fetuses designated for visceral evaluation were decapitated (head placed in appropriately labelled tissue bags and fixed in Bouin's solution for later evaluation). The fetal specimens were then secured beneath a dissecting microscope and dissected so as to permit evaluation of tissues in the thoracic and abdominal cavities.
- Skeletal examinations: Yes: half per litter; The remaining fetuses not used for soft tissue examinations in each litter were killed and the intact fetuses were eviscerated (internally sexed by inspection of the gonads) and processed for staining of the ossified skeletal structures. Fetal skeletal specimens were evaluated under a dissecting microscope (10-20X) for ossification variations and malformations.
- Head examinations: Yes: half per litter; Following a period of fixation, the fetal heads (see "soft tissue examinations" section above) were sectioned using a razor blade. The serial, traverse sections generated using this procedure were evaluated for malformation of the palate, eyes and brain under a dissecting microscope (10-20X). Following evaluation, head sections were placed in plastic cassettes for storage (one litter/jar) in a 10 % formalin solution.
Statistics:
See any other information on materials and methods including tables.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At the 540 mg/kg-bw/day dose group, 2/24 animals died (8.3% mortality), with both animals dying on day 10 of gestation. Excessive salivation was seen with increased frequency among the animals in the mid dose group during the treatment period. At the 540 mg/kg-bw/day dose group, the following findings were seen with increased frequency: lethargy, prostration, irregular gait, excessive lacrimation, labored breathing and staining of the skin/fur in the ano-genital area. Generally, these findings were most prevalent during the Day 6-10 gestation period. At the 180 mg/kg-bw/day dose group, a statistically significant reduction in food consumption was seen early in the treatment period (Days 6-9). For the remainder of the study, however, food consumption data for this group were comparable to control data. At the 540 mg/kg-bw/day dose group, food consumption was significantly lower than control throughout the treatment period. During the post treatment period food consumption of the 540 mg/kg-bw/day dose was statistically significantly increased in comparison to control data.
The mean liver to body weight ratio of the mid dose group was statistically significantly lower than controls with similar responses in liver to body weight ratio in the 540 mg/kg-bw/day dose group, this was not considered toxicologically significant.
No adverse effect of treatment was evident for maternal macroscopic postmortem examinations for any dose group. At the 540 mg/kg-bw/day dose group, 2/24 animals died for a mortality rate of 8.3%. Both animals died on day 10 of gestation. Excessive salivation was seen with increased frequency among the animals in the mid dose group during the treatment period. At the 540 mg/kg-bw/day dose high dose level, the following findings were seen with increased frequency: lethargy, prostration, irregular gait, excessive lacrimation, labored breathing and staining of the skin/fur in the ano-genital area. Generally, these findings were most prevalent during the Day 6-10 gestation period. Excessive salivation was also to the control group. Maternal body weight gains for the mid and 540 mg/kg-bw/day dose during the post-treatment period were comparable to control data with the exception of the 540 mg/kg-bw/day dose group which gained significantly more weight than control over days 16-18. Mean weight gains for the mid and high dose groups over the entire Day 6-20 gestation period using the corrected Day 20 gestation weights were lower than control and these differences were statistically significant.
As expected from the lower terminal body weights seen in the 180 and 540 mg/kg-bw/day dose groups, mean absolute liver weights for these groups were also significantly lower than controls. The mean liver to body weight ratio of the mid dose group was statistically significantly lower than controls with similar responses in liver to body weight ratio in the 540 mg/kg-bw/day dose group, this was not considered toxicologically significant. No adverse effect of treatment was evident for maternal macroscopic postmortem examinations for any dose group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
ca. 60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
ca. 180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOEL
Effect level:
ca. 540 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOEL
Effect level:
ca. 180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At the high dose level, mean fetal weights, distinguished by sex and as a composite for both sexes, were lower than control data. These differences were statistically significant and considered indicative of an adverse response to treatment. No adverse effect of treatment on fetal sex distribution data was observed for any dose level. No adverse effects of treatment were seen from fetal external examination data at any dose level. No malformations were seen during the fetal visceral examinations. A low incidence of commonly seen visceral variations was seen in the low dose group. No visceral variations were seen among the control, mid or high dose fetuses. The incidences of these findings on both a per fetus and per litter basis were well within the range of recent historical control data for the laboratory and in the absence of similar findings in fetuses from the higher dose levels, no adverse effect of treatment was indicated. No adverse effect of treatment at a dose level up to and including the mid dose was seen for the fetal skeletal malformation data. No skeletal malformations were seen in the mid or high dose fetuses. Likewise, no skeletal malformations were seen in the control fetuses. The only skeletal malformations seen were in the low dose group. The incidence of skeletal malformations in the low dose group was 2.6 % and the litter incidence was 16.7 %. These incidences did not differ statistically from control data. The incidences of fetuses with one or more ossification variations in the treated groups were comparable to or slightly lower than control data. The incidence of fetuses with at least one ossification for the low dose group differed statistically from control data but this was not considered indicative of an adverse response to treatment. The fetal and litter incidences for the different ossification variations seen during the study for the low and mid dose groups were considered comparable to control and not adversely affected by treatment. Only at the high dose was there a suggestion of retarded fetal ossification. This was indicated from the slight increases in fetal and litter incidences of reduced ossification of the cervical vertebral transverse processes and metatarsals for this group in comparison to control data.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Analytical Results:

Analyses of preliminary mixes at the low and high concentrations confirmed that the procedure used to prepare dosing solutions in this study produced homogenous mixtures and confirmed that the test substance at a concentration level of 12 mg/mL was stable in corn oil, under ambient storage conditions, for at least 14 days. Analyses conducted during the treatment period confirmed that dose solutions of appropriate concentrations were administered to the test animals. The mean analytical concentrations seen from the periodic analyses of dosing solutions used on study were as follows: 99.2 % (12.0 mg/ml), 100.6 % (36.0 mg/mL) and 102.3 % (108.0 mg/mL) of nominal.

Reprotoxicity Effects:

Pregnancy rates were comparable to controls. Actual pregnancy rates were: 91.7%, 100%, 95.8% and 95.8% for the control, 60, 180 and 540 mg/kg-bw/day dose groups, respectively. No adverse effects of treatment were evident from the number of corpora lutea, live fetuses, resorptions and uterine implantations per pregnant female. Likewise, the preimplantation loss indices and the ratio of resorptions to implants were comparable for treatment groups to the control.

Applicant's summary and conclusion

Conclusions:
Mated female rats were administered 2,6-xylenol (60, 180 and 540 mg/kg/day) via oral gavage over the Day 6-15 gestation interval. Females were sacrificed on Gestation Day 20 and the maternal toxicity and developmental toxicity of the fetuses was evaluated. Based on the results of the study, the NOEL for maternal toxicity was determined to be 60 mg/kg/day and the NOEL for developmental toxicity was 180 mg/kg/day.